eLS || Cold Acclimation and Freezing Tolerance in Plants

Cold Acclimation andFreezing Tolerance inPlantsFrancois Ouellet, Universite du Quebec a Montreal, Montreal, Quebec, Canada

Jean-Benoit Charron, McGill University, Ste-Anne-de-Bellevue, Quebec, Canada

Based in part on the previous version of this eLS article ‘Cold Acclimationand Freezing Tolerance in Plants’ (2007) by Francois Ouellet.

The capacity to survive winter conditions varies greatly in

the plant kingdom. Cold acclimation is the process lead-

ing to the development of freezing tolerance in plants. It

is a complex multigenic process that requires a pro-

grammed and integrated genetic capacity to activate the

appropriate mechanisms needed to withstand harsh

winter conditions. Hardy plants have evolved complex

mechanisms to tightly regulate gene expression, includ-

ing events at the transcriptional and post-transcriptional

levels. Hundreds of cold-induced genes encoding struc-

tural and regulatory proteins have been identified. These

proteins have been found in many species, but in most

cases, theyhad been first identified in model species. Most

of the studies in the field are still performed with the

model dicotyledonous plant Arabidopsis, but Brachypo-

dium distachyon is emerging as a model for mono-

cotyledonous species.

Introduction

Temperature and water availability are among the mostimportant environmental factors affecting plant growthand development. Because most crops of economicimportance are sensitive to temperatures less than 10 8C,significant losses can result from sudden frosts in the falland from unusual freezing temperatures in the winter.

Tolerant annual and biannual plants possess the geneticmakeup required to acquire freezing tolerance (FT)through a process called cold acclimation. This updatedversion of the authors’ 2007 review presents the progressmade in the field over the past 6 years. The authors discusswhat they feel are some of themost interesting new avenuesof research in the field. See also: Cold Acclimation andFreezing Tolerance in Plants

Cold Acclimation and FT

Low temperatures (LTs), resulting from either geographicaldistribution (latitude or altitude) or seasonal variations,limit plant growth and survival. The cold acclimation pro-cess allows hardy plants to trigger the efficient mechanismsneeded for the acquisition of FT, which determines theircapacity to survive winter. During the period of exposure toLT, numerous biochemical, physiological and metabolicfunctions are altered in plants (Thomashow, 1999). Thesechanges are regulated by LT mostly at the gene expressionlevel. In natural conditions, cold acclimation occurs in thefall, when temperatures are still above the freezing point.The maximal FT is maintained during winter, when tem-peratures are below the freezing point, and lost in the spring,when temperatures goback above the freezing point. FT is acomplex multigenic trait that requires an integrated,genetically programmed capacity to overwinter. Thismeansthat some species, andvarietieswithin each species, possess agreater acclimation capacity than others. The hardiest her-baceous species, such as winter cereals, achieve an LT50

(lethal temperature 50; temperature that kills 50% of theindividuals) of approximately –20 to –25 8C when fullyacclimated. Trees use a variety of mechanisms to withstandmuch lower freezing temperatures. In contrast, some tro-pical species will suffer irreversible damages at temperaturesjust above the freezing point.

Advanced article

Article Contents

. Introduction

. Cold Acclimation and FT

. Cold Perception and Signal Transduction

. Plasma Membrane Modifications

. Photosynthesis and Sugar Metabolism

. Modifications in Gene Expression

. Structural Proteins

. Promoters and Transcription Factors

. Influence of Trans Regulators on LT-Responsive Gene

Expression

. Regulation of Gene Expression by Small RNAs

. Chromatin Regulation

. Brachypodium distachyon as a Model Organism

Online posting date: 9th December 2013

eLS subject area: Plant Science

How to cite:Ouellet, Francois; and Charron, Jean-Benoit (December 2013) ColdAcclimation and Freezing Tolerance in Plants. In: eLS. John Wiley &

Sons, Ltd: Chichester.

DOI: 10.1002/9780470015902.a0020093.pub2

eLS & 2013, John Wiley & Sons, Ltd. www.els.net 1

http://dx.doi.org/10.1002/9780470015902.a0020093

http://dx.doi.org/10.1002/9780470015902.a0020093

As temperatures fall, ice forms in the apoplastic space andcytoplasmic water is drawn from the cells to the growingmass of extracellular ice. To avoid the loss of water, cells offrost-tolerant organisms accumulate low molecular weightsolutes, such as proline, sugars and glycinebetaine (Burg andFerraris, 2008; McNeil et al., 1999). This occurs because LTinduces the expression of genes encoding enzymes requiredfor the biosynthesis of these compounds, such as d-1-pyr-roline-5-carboxylate synthetase, phosphoethanolaminemethyltransferase, betainealdehyde dehydrogenase, sucrosesynthase, galactinol synthase and others. Interestingly, someof the corresponding genes are part of the C-box bindingfactor (CBF) regulon, the most studied cold-responsivepathway in plants (see section ‘Promoters andTranscriptionFactors’). The compatible solutes contribute to the increasein osmotic potential and concomitant decrease of the cyto-plasmic freezing point. The latter property prevents theformation of deleterious ice crystals that would cause irre-versible structural damage and lead to cellular death. Thesolutes also stabilise the structure of cell membranes andproteins, thus preserving their functional capacities follow-ing a freeze–thaw cycle. In addition, several proteins withantifreeze properties participate in cellular protection (seesection ‘Structural Proteins’). The genes that are induced byLTare collectively referred toas cold regulated (COR)genes.The improvement of cold tolerance by traditional

breeding depends on the availability of species/varietiesthat inherently show high levels of cold tolerance.Although traditional breeding approaches have generateda remarkable improvement in the cold tolerance of certainplants, extensive breeding has caused the depletion of FT-associated genes available in the genetic pools of eachspecies. Thismeans thatmost of the genes that are involvedin the protection mechanisms have been exploited, so fur-ther improvement is very limited and labour intensive.The identification of FT-associated genes can lead to thedevelopment of molecular markers that will assist thebreeders in the selection of plants showing the best genetic

makeup for the desired FT trait, hence greatly improvingthe breeding efforts to obtain better cultivars. However,genetic manipulation offers the possibility of crossingspecies barriers and allows the transfer of tolerance genesidentified in cold-tolerant species to cold-sensitive speciesof any family. To establish a good improvement strategy,and because FT is a multigenic trait, it is essential tounderstand the mechanisms involved in the cold acclima-tion process and development of FT. This involves study-ing the different aspects associated with the adaptivemechanisms of tolerant species. In addition, becausefreezing stress shares some common characteristics withother stresses, such as drought and salinity, it is importantto distinguish the events that are specific to FT from thoserelated to a general stress response. The physiological andbiochemical modifications resulting from LT exposurehave been described extensively (Thomashow, 1999). Lessunderstood is how the LT stimulus is perceived by the cells,how the signal is transduced andwhat specific elements arerequired for proper expression of cold-inducible genes.Over the past 25 years, a considerable and increasinginterest in the field has led to major breakthroughs in ourunderstanding of LT perception and induction of LT-responsive gene expression (Figure 1). See also: Plant StressPhysiology; Plant Temperature Stress

Cold Perception and SignalTransduction

Cold acclimation and the development of FT requireproper perception, precise signalling and a coordinatedand tightly controlled regulation of gene expression. Themechanisms by which plant cells perceive the LT stimulusare poorly understood. The plasma membrane is recog-nised as the primary site of injury and is hypothesised toalso be a site of perception of the LT stimulus (Uemura

Activation ofLT-responsive genes

Signal detection andtransduction pathway

Induction of freezingtolerance

Light

Chloroplast?Metabolic shiftsCarbohydrates, lipids,proteins, amino acids,hormones and cellularenergy

Receptor ?Cold

IntracellularsignalCa2+, proteinphosphorylation

StructuralreorganisationPlasma membrane,cytoskeleton organelles andprotein synthesizingmachinery

DNA RNA Proteins

Nucleus

?

Cytosol

Figure 1 Cellular events associated with the development of FT in plants. Cold is perceived at the plasma membrane by an unknown mechanism that

might depend on physical modifications and by the chloroplasts in terms of photosynthetic activity. Various signal transduction cascades are triggered,

some involving calcium and phosphorylation events. Gene expression is modified and the corresponding proteins adjust the cell’s structure and metabolism.

Reproduced from Ouellet (2002) with permission of Society for In Vitro Biology, formerly the Tissue Culture Association. & Society for In Vitro Biology.

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Cold Acclimation and Freezing Tolerance in Plants

http://dx.doi.org/10.1038/npg.els.0001297



et al., 2006). Putative cold sensors include the actin cytos-keleton, via modifications in plasma membrane physicalproperties, and the chloroplast, via modifications in thephotosynthetic efficiency. The LT stimulus is transducedvia both abscisic acid (ABA)-dependent and -independentpathways, with calcium being an important secondarymessenger. The calcium signal is more likely transduced bya series of phosphorylation events that would involve cal-cium-dependent protein kinases. Although several cal-cium-dependent and -independent protein kinases havebeen cloned to date, there is little information regarding thespecific downstream signalling components leading to theactivation of specific subsets of genes. A major difficultylies in the fact that many LT-responsive genes are alsoinduced by other stimuli, such as drought, ABA and sali-nity, and it is still unclear how these different stimuli con-verge to induce the expression of the same genes. ExposuretoLT leads to a loss ofwater and the production of reactiveoxygen species (ROS). In fact, abiotic stresses that causecellular dehydration or ROS accumulation, such as LT,drought and salinity, induce common subsets of genes,indicating that common responses are required to developtolerance to these stresses. It is believed that ROS wouldhave a role in plant sensing and signalling of the tempera-ture stimulus (Suzuki et al., 2012). Maintaining or con-trolling the levels of ROS is critical to prevent detrimentalconsequences. The superoxide produced by nicotinamideadenine dinucleotide phosphate-oxidases during LTexposure activates stress response pathways and inducesdefence mechanisms. This occurs via the activation of anoxidative stress regulon in plants, and it is thought that thisregulon overlaps with the different networks controllingtemperature stress acclimation and tolerance. Anti-oxidants, such as glutathione, ascorbate and Vitamin E,and enzymes involved in detoxification of ROS, such ascatalase and superoxide dismutase, have been suggested toprotect plants against freeze–thaw damage.Evidence is accumulating for the involvement of a

phosphoinositides transduction pathway in abiotic stresssignalling (Chinnusamy et al., 2010). An inositol poly-phosphate 1-phosphatase, FIERY1, regulates cytosolicinositol-1,4,5-triphosphate levels, a molecule that cantrigger calcium release from intracellular stores. Otherstudies have provided evidence for the involvement of amitogen-activated protein kinases (MAPK) pathway(AtMEKK1–AtMKK2–AtMPK4/6) in LT and otherabiotic stress signal transduction. The phosphoinositidesand MAPK pathways have first been described in animalsystems; therefore, evidence has now clearly establishedthat the plant and animal kingdoms share many similarcharacteristics in their signal transduction pathways.

Plasma Membrane Modifications

The plasma membrane is considered to be the primary siteof freezing injury in plant cells (Steponkus, 1984). Its lipidcontent has been extensively studied. Both the relative

abundance and saturation level ofmembrane lipids changefollowing exposure to LT. The general desaturation ofthese lipids ensures that themembranes will remain as fluidas possible when temperatures drop. Plants that respondbetter at the molecular level to express and activate theenzymes required for desaturation or hydrolysis of fattyacids have a better ability to cold acclimate. If thisadjustment does not take place as temperature is decreased,significant structural damages occur when cell membranesundergo phase transition from a liquid crystalline to a gelstate. This adjustment is important because the physicalstructure (gel or fluid phase) of lipid domains in mem-branes influences cellular signalling and other biologicalfunctions. If the temperature at which the membrane lipidphase transition occurs could be lowered, or if the mem-brane phase transition could be eliminated completely,membranes would remain fluid at LTs and membranedamage might be reduced. In addition to lipids, proteinsare also more likely to play a role in the stabilisation ofmembranes. Proteomics analyses are the latest technolo-gies used to study changes that occur in the plasma mem-brane during acclimation and freezing (Uemura et al.,2006). One of the main findings from these studies is thatFT-associated proteins, such as dehydrins and lipocalins,accumulate at the plasma membrane during LT exposure.Although their precise mode of action is unknown, evi-dence suggests that dehydrins and lipocalins protectmolecules and structures against freeze-induced damageand reduce the extent of oxidative stress, respectively.

Photosynthesis and SugarMetabolism

Higher plants subjected to changing environmental con-ditions, such as LT, have to adjust their photosyntheticcapacity (Huner et al., 2013). This involves adjusting thechloroplastic antennas, which are composed of pigments(mostly carotenoids) and proteins. As there is a delicateequilibrium between the energy that is harvested and theenergy that is used, it has been suggested that the redoxstatus of photosynthesis could be an important signallingmechanism, especially during cold stress. Exposure ofplants to LT may induce high excitation pressure andcreate an energy imbalance. However, photosyntheticorganisms have evolved a number of mechanisms toimprove such conditions. Chronic exposure to high exci-tation pressure may lead to photoinhibition, which is alight-dependent decrease in photosynthetic rate that occurswhen the photon flux is in excess. This excess light can beeliminated by thermal dissipation through a processknown as nonphotochemical quenching, and plants thatcan cold acclimate have more efficient mechanisms to doso. If the absorbed energy exceeds both the photochemicaland nonphotochemical quenching capacities, the result isirreversible photoinhibition or photodamage. Damages tothe chloroplast structure disrupt its photosynthetic



capacity. It has been suggested that photosynthesis couldmore likely function as a sensor of this imbalance throughthe redox state of photosynthetic electron-transport com-ponents, resulting in the regulation of photochemical andmetabolic processes in the chloroplast. Because light isessential for the development ofmaximal FT, itmore likelyappears that photosynthesis, and perhaps other processestaking place in the chloroplast, is crucial to cold acclima-tion and FT. An efficient communication channel must,therefore, exist between the chloroplast and the nucleus toensure the proper metabolic adjustment needed for thedevelopment of FT. This retrograde signalling is known tooccur during drought stress, which shares componentssimilar to LT stress (Chan et al., 2010).

Another means to adjust photosynthetic efficiency is therelocation of chloroplasts when plants are exposed tostresses, including LT (aka chloroplast cold positioning).This movement is dependent on both LT and light and hasbeen known to occur in plants for a century. In liverwortplants grown under these conditions, chloroplasts willmove from areas of high light intensity to those of lowerintensities (from one side of the cell to the other) to mini-mise photodamage (Ogasawara et al., 2013). In addition,this study has shown that nuclei and peroxisomes, but notmitochondria, also respond to cold positioning. The bio-chemical relevance of thismovement is currently unknown.In addition to the adjustment of the photosynthetic

apparatus, it was suggested that sugar-signalling pathwayscould regulate plant acclimation to LT. What is known isthat overwintering plants accumulate higher levels ofsoluble carbohydrates, such as sucrose, glucose, fructose,stachyose, raffinose, sorbitol and mannitol (Kaplan et al.,2007). Some of these sugars increase the intracellularosmolytes concentration and thus lower the freezing pointof the cytoplasm. In the chloroplast, starch is hydrolysedby b-amylase to produce the cryoprotective compoundmaltose, a precursor of soluble sugar metabolism. Thetranscript levels and activity of the different amylase iso-forms are regulated by environmental stimuli, such as cold,heat and drought stress. The activity of the chloroplasticisoform in Arabidopsis is needed for the protection ofphotosystem II efficiency following freezing stress, pre-sumably because it produces maltose.

Modifications in Gene Expression

LT leads to important modifications in gene expression. Itis estimated that the expression of nearly 10%of all genes isaffected, one way or another, by exposure to cold tem-peratures. Thismodification of the transcriptome results inimportant genetic,metabolic and structural adjustments atthe cellular level. A plethora of genes are expressed inresponse to LT, and the molecular responses associatedwith the induction of their expression are complex. Large-scale expressed sequence tags analyses revealed that thestress response is partly conserved between wheat andArabidopsis, two phylogenetically distant plant species

(Houde et al., 2006; Laudencia-Chingcuanco et al., 2011).More than 44%of the 2637 putativewheat stress-regulatedgenes have a homologue that is regulated by stress inArabidopsis (Houde et al., 2006). A comprehensive dis-cussion of all the proteins associated with cold response isbeyond the scope of this article. The authors here reviewthe best-known LT-associated proteins and how LT reg-ulates gene expression at the molecular level.

Structural Proteins

Dehydrins are the most conspicuous subset of solubleproteins induced by dehydrative stresses, such as LT,drought and salinity stress. They have a broad size range,show no significant similarity in amino acid sequence toproteins of known function and accumulate to levels inexcess of 1% of total soluble proteins. Dehydrins can beclassified by their content in different amino acid segmentsnamed K, S and Y, which are usually repeated severaltimes. Dehydrins are also peculiar by their high hydro-philicity and boiling solubility, which facilitate their pur-ification. Their widespread cellular distribution andpropensity to adopt an a-helical structure in the presence ofSDS is consistent with a role in the protection of cellularstructures and molecules against freezing-induced dama-ges. It was shown that dehydrins can protect enzymes fromfreezing damage in vitro. For example, the wheat WCS120protein at 0.2 mM is as efficient as 250mM sucrose in thecryoprotection of lactate dehydrogenase (more than amillionfold more efficient on a molar basis). First, it wasproposed that dehydrins, acting synergistically with com-patible solutes, may replace water for the ‘solvation’ ofmembranes and proteins. This propertywould alleviate thedehydration-induced membrane destabilisation and pro-tein coagulation. Second, these proteins may reduce theincidence of events leading to the formation of nonbilayerstructures between membranes. Finally, dehydrins maycounteract the irreversible damages of increasing con-centrations of various ions in the cytoplasm during dehy-dration. High ionic concentrations decrease interbilayerrepulsiondue to charge screening and lead to an interactionbetween bilayers and the induction of nonbilayer struc-tures. It is not surprising that dehydrins have been found inmost plant species exposed to cold temperatures studied sofar, irrespective of whether they show a good FT or not.Intriguingly, overexpression of dehydrins in Arabidopsisand other plants only resulted in modest increases in FT.This suggests that even though these proteins accumulateduring LT exposure, they may not be the major determi-nants of tolerance.Another LT-responsive gene family encodes antifreeze

proteins (AFPs) (Griffith and Yaish, 2004). These proteinshave a high affinity for ice and possess two typical prop-erties: ice recrystallisation inhibition and thermal hyster-esis (the difference between the freezing and the meltingpoints). They accumulate at high concentrations in theapoplast, the extracellular space between the plasma



membrane and the cell wall, where they bind ice crystals.The coating of the crystals restricts their growth, therebyenabling plants to survive freezing conditions. Interest-ingly, some AFPs show homology to a group of plantpathogenesis-related proteins that includes b-1,3-gluca-nases, endochitinases and osmotin- and thaumatin-likeproteins. Other AFPs, like the wheat TaIRI ice recrys-tallisation inhibition proteins, have a peculiar bipartitestructure. They showa leucine-rich repeat receptor domainof receptor-like protein kinases at theirN-terminus and icerecrystallisation inhibition domains at their C-terminus(Tremblay et al., 2005). The available data on the char-acterisation of the different AFPs suggest that they wouldhave a role in the tolerance of plants to freezing stress bypreventing the formation of large ice crystals that coulddamage cellular structures. The homology of some AFPswith disease resistance proteins raises the possibility of adual role in abiotic and biotic stress tolerance.

Promoters and Transcription Factors

TheLT-inducible accumulation of transcripts and proteinsindicated the existence of regulatory elements controllinggene expression at the transcriptional level. A major focusof research aimed at the understanding of LT-inducedmodifications in gene expression has thus been directedtowards the identification of cis-acting promoter elementsand transcription factors binding them. Despite the factthat many groups have studied promoters of LT-respon-sive genes in various plant species, only one cis-regulatoryelement (and close variants) has been identified as a gen-uine LT-responsive element (LTRE) and shows the con-sensus sequence RCCGAC (where R is a purine, A or G).The transcription factors binding this element, namedCBF, were first identified in Arabidopsis. They belong to asubset of the AP2 class of transcription factors and havebeen found in most plant species examined so far. Over-expression of CBF genes in Arabidopsis increases FTwithout any requirement for LT exposure, indicating thatthis transcription factor is a major player in the develop-ment of FT. Following a rapid induction of CBF transcriptaccumulation in response to LT, the corresponding pro-teins accumulate and induce the subsequent accumulationof their target genes, which invariably contain LTREs intheir promoters. Large-scale microarray and genomicstudies later identified the CBF ‘regulon’, the set of genesup- or downregulated by CBFs (Carvallo et al., 2011).Because the accumulation of CBF transcripts occurs assoon as plants are exposed to LT, it was speculated thatconstitutively present transcription factors regulating theirexpression must exist. Elegant studies performed usingCBF3 promoter-driven luciferase expression allowed theidentification of inducer of CBF expression 1 (ICE1)(Chinnusamy et al., 2003). This basic helix–loop–helixtranscription factor is constitutively expressed and reg-ulates nearly 1000 downstream targets, including CBF3and other cold-induced regulons (Lee et al., 2005).

Consequently, it has a dramatic effect on the developmentof FT. The level of ICE1 protein is regulated by the variantRING finger protein HIGH EXPRESSION OF OSMO-TICALLY RESPONSIVE GENE 1 (HOS1), a negativeregulator of cold responses. LT exposure induces the ubi-quitination and subsequent proteolysis of ICE1, reducingthe accumulation of CBF and delaying the establishmentof FT.Other transcription factors are also involved in cold

response. Data generated by transcriptomics analysesperformed in Arabidopsis indicated that many cold-responsive genes are part of the CBF regulon. However,several cold-responsive genes are not regulated by the CBFtranscription factors, including genes that encode knownor putative transcription factors (Vogel et al., 2005). Theseresults support the existence of both CBF-dependent and-independent regulatory pathways associated with LTresponse in Arabidopsis. One of the latter pathwaysinvolvesZAT12, a zinc-finger protein predicted to encode atranscriptional repressor. Overexpression of ZAT12represses genes that are negatively associated with LT andinduces the expression of genes that are positively asso-ciated with LT. The overall effect should have been a fairlygood increase inFTof nonacclimated plants.However, theobserved improvement was modest, indicating that thispathway is not as important as the CBF pathway for thedevelopment of FT. Interestingly, there is an overlap in theregulons affected in the ZAT12 and CBF cold responsepathways, indicating that they crosstalk. Seven genes forwhich the expression is affected by LT exposure are part ofboth the CBF and ZAT12 regulons. Other studies revealedthat the transcription factors HOS9 (a homoeodomaintype) and HOS10 (a R2R3myeloblastosis type) are part ofother CBF-independent pathways of gene regulation inresponse to LT.Although global, genomic-scale technologies helped to

delimit cold stress-related regulons, the signalling eventsfrom sensors (perception) to transcription factors to cel-lular responses require further investigation. Future stu-dies need to be focussed on determining the genes that arethe major factors regulating FT. One aspect that is over-looked and which should be investigated is the role playedby genes that are downregulated by exposure to LT.

Influence of Trans Regulators on LT-Responsive Gene Expression

Vernalisation is the promotion of flowering by a coldtreatment, a physiological process that has been best stu-died in monocotyledonous winter growth habitGramineaespecies. Because a plant that has achieved its vernalisationrequirement loses its FT, it was suggested that there waslikely a relationship between the responses related to ver-nalisation and those related to cold tolerance. Spring habitcereal cultivars do not have a vernalisation response andare unable to maintain LT-induced gene expression in an



upregulated conditionwhen exposed to LT.Consequently,they are unable to achieve levels of FT similar to those ofwinter cultivars. The close association between the point ofvernalisation saturation and the start of a decline in mes-senger ribonucleic acid (mRNA) and protein levels of FT-associated proteins, such as the wheat WCS120 dehydrin,indicates that vernalisation genes play a key role in deter-mining the duration of expression of the FT-associatedgenes (Fowler et al., 1996). This suggests an explanation forthe apparent pleiotropic effect that the VRN-A1 (vernali-sation) locus has on both the vernalisation response andFT in wheat. It appears that any factor that delays thetransition from vegetative to reproductive stages, such as avernalisation or photoperiod requirement for flowering,would be expected to increase the level of expression of FTgenes in cereals exposed to acclimating temperatures. TheVRN-A1 locus maps to the long arm of chromosome 5A,the same chromosome that was shown to bear factorshaving a positive influence on the regulation of LT-indu-cible genes. The corresponding gene, TaVRN1, encodes aMADS-box transcription factor that positively regulatesthe transition from the vegetative to reproductive phase(Danyluk et al., 2003). In winter wheat, at least two othertranscription factors are important for the negative reg-ulation of flowering. The MADS-box TaVRT2 and thezinc-finger TaVRN2 proteins act by delaying the accu-mulation of TaVRN1. The two repressors accumulateduring the vegetative phase in winter cereal varieties.TaVRT2 directly binds its target element, called a CArGbox, in the TaVRN1 promoter. The binding represses theactivity of the promoter, resulting in a lower accumulationof the TaVRN1mRNA and protein. This repression effectis enhanced or stabilised by TaVRN2. During vernalisa-tion, TaVRT2 and TaVRN2 transcript levels decline by amechanism that is still unknown, and this allows the gra-dual accumulation of TaVRN1 and subsequent transitionfromvegetative to reproductive phase. Spring growthhabitcereal species do not express the TaVRT2 and TaVRN2repressors; therefore, they show a high level of TaVRN1protein from their early development until the flowers aremature. Intriguingly, it was reported that contrary to whatwas observed for wheat, the expression of VRT2 increasesduring LT treatment in barley, and the authors concludethat VRT is unlikely to regulate VRN1 (Trevaskis et al.,2007). This discrepancy between the two systems requiresfurther investigation. Another aspect that needs to bestudied is how TaVRN1 exerts its effect on the accumula-tion of LT-inducible transcripts, such as the dehydrins.

Regulation of Gene Expression bySmall RNAs

There has been a considerable interest in the study of smallRNAs over the past 10 years. It has been suggested that atleast 30% of all genes are regulated at the post-transcrip-tional level by these RNAs, but this number is more likely

to be higher as large-scale studies progress.Most, if not all,of the physiological processes during normal developmentor in response to environmental stresses depend on smallRNAs regulation of gene expression (Sunkar et al., 2007).The two main classes of small RNAs are the microRNAs(miRNAs) and the small interfering RNAs (siRNAs). ThemiRNAs and a subclass of siRNAs are 21–24 nucleotidesin length. The genes encoding these RNAs are distributedin the genomeand the current understanding is thatmost ofthem are at loci that are distinct from those encoding theirtarget genes. This implies that they bear their own pro-moter region and therefore they have their own transcrip-tional regulation. As for most transcripts, the primarytranscripts are capped at their 5’ end and polyadenylated attheir 3’ end (reviewed in Rogers and Chen (2013) andZhang et al. (2012)). The involvement of miRNAs in post-transcriptional regulation of LT-inducible gene expressionhas been demonstrated (Tang et al., 2012; Zhou et al.,2008). Because miRNAs decrease the level of their targetRNAs, the LT regulation of their expression will have adirect consequence on the levels of their targets (Sunkaret al., 2007). This is an exciting new field that promises toreveal several new aspects of LT gene regulation, and itmight lead to the discovery of yet unidentified physiologi-cal and metabolic adjustments that occur when plants aresubjected to LT.

Chromatin Regulation

Several studies have recently established that cis-actingelements and trans-acting transcription factors are not theonly transcriptional regulatory mechanisms used byeukaryotes to respond to adverse environmental condi-tions, and evidence of a positive role for chromatin ontranscription has been established (Thomas and Elgin,1988). Chromatin is a highly organised structure built ofrepeating nucleosome subunits that not only allows thepackaging of large quantities of genetic material into aminute nucleus but also serves as an efficient integrativeplatform that translates diverse signals from the cellularenvironment into orchestrated responses from deoxyr-ibonucleic acid (DNA) (Knizewski et al., 2008). Thechromatin state is dynamic and tightly modulated to eitheran ‘open’ (transcriptionally active) or a ‘closed’ (tran-scriptionally repressed) configuration by the local action ofmultiple regulators/modifiers. These modifiers areresponsible for adding or removing covalent modificationsfrom histones and DNA and for regulating nucleosomescomposition, density and positioning. One of the bestcharacterised modifications is histone acetylation. Whenthe nucleosome histones are not acetylated, the DNA istightly wrapped around the nucleosome core and the geneslocated in these compacted areas are poorly accessible forthe transcriptional machinery. However, when acetylationoccurs, the chromatin state changes to a relaxed con-formation that allows amore efficient transcription at theseloci. In addition, somemodifiers interactwith small RNAs,



which have emerged as important regulatory componentsof the chromatinmodification system (Steimer et al., 2004).

Despite the general understanding of eukaryotic chro-matin structure, very few links between the regulation ofcold stress response and chromatin dynamics have beenidentified in plants so far. Early evidence implicated agroup of chromatin modifiers involved in histone acetyla-tion (Figure 2). In vitro studies demonstrated that the HATGCN5 is recruited by the CBF1 transcription factorthrough the transcriptional adaptor ADA2 to induce theexpression of target COR genes (Stockinger et al., 2001).Mutations in GCN5 and ADA2 hinder the capacity ofArabidopsis to express cold-responsive genes, which nega-tively impacts their overall level of FT (Vlachonasios et al.,2003). The working hypothesis at that point was relativelysimple: under cold conditions, the protein complex com-posed ofGNC5,ADA2 andCBF1would target promotersof COR genes and bring about changes in the acetylationstatus of nucleosomes. The chromatin state would thuschange to a more relaxed conformation and allow a moreefficient transcription of COR genes.This working model found further support when con-

stitutive H3 acetylation levels and decreased nucleosomeoccupancy were observed at the promoters of COR genesin Arabidopsis plants overexpressing AtCBF1 (Pavangad-kar et al., 2010). However, the same study provided con-flicting results by suggesting that neither ADA2b norGCN5 is required for histone acetylation at COR genepromoters of cold-treated plants, clearly indicating that

GCN5 is not the only HAT responsible for acetylatingnucleosomes at COR gene promoters. This assumptionwas further confirmed when certain level of redundancyamongst HATs was revealed, as several HAT Arabidopsismutants did not demonstrate impaired acetylationpatternsat several COR gene promoter regions. However, a moredirect role for GCN5 and ADA2 in cold acclimation wasput forward asGCN5 is thus far the onlyHAT shown to becapable of clearing nucleosomes at COR gene promotersunder cold conditions. This could represent the majorcontributionofArabidopsisADA2bandGCN5proteins tothe cold acclimation process rather than simply the directaddition of acetyl groups to histone proteins (Pavangadkaret al., 2010).

The impact of HDACs on cold-inducible gene expres-sion has not been extensively studied and is much lessunderstood. Genetic analysis indicated that ArabidopsisHOS15 is involved in deacetylating histones at the pro-moter of the COR gene RD29A (Zhu et al., 2008). HOS15shares homology with a component of the human SMRT/N-CoR gene repressor complex that is involved in mod-ification of chromatin structure by its associated HDACs.HOS15, a WD40 protein, was also shown to interactdirectly with histone H4 and to function as a transcrip-tional repressor (Zhu et al., 2008). Plants deficient inHOS15 are more sensitive to freezing temperatures anddisplay higher than normal levels of histone acetylation.The involvement of HOS15 in cold tolerance suggests thatthe dynamic balance between histone acetylation of

20 °C 4 °C

Positive effectors(e.g. CBF1)

Negative effectors

HDACs HAT

ACAC

ACACAC

AC

Positive effectors(e.g. CBF1)

Negative effectors

HAT HDAC

ACAC

ACAC

AC

AC

COR

HDAC

COR

ADA2

CBF1

GCN5HAT

AC

ACAC

AC

Nucleosome clearing

Figure 2 Model showing the relationship between dynamic changes in histone acetylation levels and the cold acclimation status of a plant. A dynamic

balance between histone acetylation (AC) by histone acetyltransferases (HATs) and deacetylation by histone deacetylases (HDACs) determines the

expression of cold-regulated (COR) genes and thus the cold response status of the plant. Under normal growth conditions, HDACs target nucleosomes

(circles) surrounding transcription start sites (TSSs) of CBF genes and other positive effectors, restricting their expression. Concomitantly, negative effectors

are targeted by HATs. This results in the inhibition of COR gene expression at 20 8C. On perception of the low-temperature signal (4 8C), HATs and HDACs

target nucleosomes of positive and negative effectors, respectively. In addition, HATs directly acetylate nucleosomes surrounding the TSSs of COR genes.

Additional evidence suggests that the GCN5 HAT is capable of clearing nucleosomes at the TSSs of COR genes. GCN5 is recruited by the CBF1 transcription

factor through the transcriptional adaptor ADA2 to stimulate the expression of target COR genes. The overall effect is an induction of COR gene expression

at LT, leading to increased FT.



positive effectors and deacetylation of negative effectors ofcold tolerance contributes to the determination of the coldresponse status of the plant.In addition, LThas been reported to induceHDACs and

to trigger extensive deacetylation of histones H3 andH4 inmaize plants (Hu et al., 2011). Treatment with the HDACinhibitor trichostatin A under low-temperature conditionsstrongly reduces the expression of the maize COR genesZmDREB1 and ZmCOR413, whereas the induction ofdrought and salt-inducible genes remains largely unaf-fected. This suggests that HDACs can positively regulatethe expression of some COR genes, more likely throughhistone deacetylation at negative regulator loci, and thatthis COR gene activation is both gene- and site-specific.Histone acetylation is not the only covalent histone

modification that has been shown to be involved in CORgene regulation. A recent study monitoring the level of thewell-characterised repressive histone methylation markH3K27me3 at two COR gene loci, COR15A andATGOLS3, revealed the presence of this mark on tran-scriptionally inactive COR genes and a progressive deple-tion after LT activation of these genes (Kwon et al., 2009).Following deacclimation, the expression of these genesreturned to normal without proper restoration at bothpromoters of the H3K27me3 mark levels, thus providingsupport for the use of this histone mark as a potential coldstress memory marker (Kwon et al., 2009).

Nucleosome remodelling is reported to be a key com-ponent for temperature perception in plants. Recent stu-dies in both Arabidopsis and Brachypodium distachyonrevealed that the occupancy of nucleosomes containing thealternative histone H2A.Z at thermally responsive genes isreduced as temperature increases and that these nucleo-somes are essential to the precise perception of the ambienttemperature (Boden et al., 2013; Kumar andWigge, 2010).Although thismechanismhas been shown to be involved attemperatures ranging from 12 to 27 8C, it remains to beseen if similar mechanisms are involved at temperaturescloser to the freezing point.

Brachypodium distachyon as a ModelOrganism

Mono- and dicotyledonous species differ in their geneticinformation and in several of their physiological responses.Although the dicot Arabidopsis is accepted as the quintes-sential plant model system, it is in fact not the best modelfor some processes taking place in the monocots. This isparticularly true for FT and vernalisation. In Arabidopsis,a limited FT is achieved after cold acclimation (approxi-mately 28 8C) compared with the high FT seen in wintercereals (approximately 223 8C), and there is not a propervernalisation response. These differences warrant theestablishment of a monocot model system that is bettersuited for the study of LT responses.

Brachypodium distachyon (L.) (Purple false brome) hasbeen introduced as a newmodel system for grass genomicsand gene discovery. It possesses traits desired in a modelplant, including a small genome, availability of diploidecotypes, self-fertility, ease of transformation, small sta-ture and a relatively rapid life cycle. In addition, genomicsequence data indicate that the Brachypodium genomeexhibits a high level of colinearity and synteny with gen-omes of the temperate cereal grains that are of major eco-nomic importance, such as wheat and barley (TheInternational Brachypodium Initiative, 2010). Approxi-mately 250Brachypodium diploid and polyploid accessionsthat show phenotypic variations in many traits of agri-culture relevance in crops are available from differentsources (Filiz et al., 2009; Vogel et al., 2009). These acces-sions reflect the numerous environments in which Bra-chypodium is most commonly found, which denotes a widerange of biotic and abiotic adaptive natural genetic varia-tions that facilitate functional genomics studies (TheInternational Brachypodium Initiative, 2010). Thus, it isnot surprising that the Brachypodium model has alreadyprovided valuable information in a number of biotic andabiotic stress tolerance studies (Luo et al., 2011; Peraldiet al., 2011; Schwartz et al., 2010). However, little is knownabout the capacity of Brachypodium to cold acclimate anddevelop FT. The first indication that cold responsemechanisms could be present in Brachypodium was pro-vided in 2010, when high-throughput sequencing andgenome-wide data mining identified 3 conserved miRNAsand 25 predicted miRNAs that show significantly higherlevels in response to cold stress (Zhang et al., 2009). Arecent study demonstrated that Brachypodium possessesthe molecular circuitry required to activate COR geneexpression (Li et al., 2012). Finally, the transcriptome ofvernalised versus nonvernalised Brachypodium was char-acterised through the use of RNA-sequencing analysis,which revealed that the expression of 1665 different genesin the Brachypodium genome is differentially regulated byat least fourfold in response to a low-temperature treat-ment (Huan et al., 2013). Together, these studies clearlydemonstrate the potential of Brachypodium in under-standing the mechanisms of cold hardiness in monocots.

References

Boden SA, Kavanova M, Finnegan J and Wigge PA (2013)

Thermal stress effects on grain yield in Brachypodium dis-

tachyon occur via H2A.Z-nucleosomes. Genome Biology 14(6):

R65.

BurgMB and Ferraris JD (2008) Intracellular organic osmolytes:

function and regulation. Journal of Biological Chemistry

283(12): 7309–7313.

CarvalloMA,PinoMT, JeknicZ et al. (2011)Acomparison of the

low temperature transcriptomes and CBF regulons of three

plant species that differ in freezing tolerance: Solanum com-

mersonii,Solanum tuberosum, andArabidopsis thaliana. Journal

of Experimental Botany 62(11): 3807–3819.



Chan KX, Crisp PA, Estavillo GM and Pogson BJ (2010)

Chloroplast-to-nucleus communication: current knowledge,

experimental strategies and relationship to drought stress sig-

naling. Plant Signaling & Behavior 5(12): 1575–1582.

Chinnusamy V, OhtaM, Kanrar S et al. (2003) ICE1: a regulator

of cold-induced transcriptome and freezing tolerance in Ara-

bidopsis. Genes & Development 17(8): 1043–1054.

Chinnusamy V, Zhu JK and Sunkar R (2010) Gene regulation

during cold stress acclimation in plants. Methods in Molecular

Biology 639: 39–55.

Danyluk J, KaneNA, BretonG et al. (2003) TaVRT-1, a putative

transcription factor associated with vegetative to reproductive

transition in cereals. Plant Physiology 132(4): 1849–1860.

Filiz E, Ozdemir BS, Budak F et al. (2009) Molecular, morpho-

logical, and cytological analysis of diverse Brachypodium dis-

tachyon inbred lines. Genome 52(10): 876–890.

Fowler DB, Chauvin LP, Limin AE and Sarhan F (1996) The

regulatory role of vernalization in the expression of low-tem-

perature-induced genes in wheat and rye. Theoretical and

Applied Genetics 93(4): 554–559.

Griffith M and Yaish MWF (2004) Antifreeze proteins in over-

wintering plants: a tale of two activities.Trends in Plant Science

9(8): 399–405.

HoudeM,BelcaidM,Ouellet F et al. (2006)Wheat EST resources

for functional genomics of abiotic stress.BMCGenomics 7: 149.

Hu Y, Zhang L, Zhao L et al. (2011) Trichostatin A selectively

suppresses the cold-induced transcription of the ZmDREB1

gene in maize. PLoS One 6(7): e22132.

Huan Q, Mao Z, Zhang J, Xu Y and Chong K (2013) Tran-

scriptome-wide analysis of vernalization reveals conserved and

species-specific mechanisms in Brachypodium. Journal of Inte-

grative Plant Biology 55(8): 696–709.

Huner NPA, Bode R, Dahal K et al. (2013) Shedding some light

on cold acclimation, cold adaptation, and phenotypic plasti-

city. Botany 91(3): 127–136.

KaplanF,KopkaJ, SungDY et al. (2007)Transcript andmetabolite

profiling during cold acclimation of Arabidopsis reveals an intri-

cate relationship of cold-regulated gene expression with mod-

ifications in metabolite content. Plant Journal 50(6): 967–981.

Knizewski L, Ginalski K and Jerzmanowski A (2008) Snf2 pro-

teins in plants: gene silencing and beyond. Trends in Plant Sci-

ence 13(10): 557–565.

Kumar SV andWigge PA (2010) H2A.Z-containing nucleosomes

mediate the thermosensory response inArabidopsis.Cell 140(1):

136–147.

Kwon CS, Lee D, Choi G and Chung WI (2009) Histone occu-

pancy-dependent and -independent removal of H3K27 tri-

methylation at cold-responsive genes in Arabidopsis. Plant

Journal 60(1): 112–121.

Laudencia-Chingcuanco D, Ganeshan S, You F et al. (2011)

Genome-wide gene expression analysis supports a develop-

mental model of low temperature tolerance gene regulation in

wheat (Triticum aestivum L.). BMC Genomics 12: 299.

Lee BH,HendersonDAandZhu JK (2005) TheArabidopsis cold-

responsive transcriptome and its regulation by ICE1.Plant Cell

17(11): 3155–3175.

Li C, Rudi H, Stockinger EJ et al. (2012) Comparative analyses

reveal potential uses ofBrachypodium distachyon as amodel for

cold stress responses in temperate grasses. BMC Plant Biology

12: 65.

Luo N, Liu J, Yu X and Jiang Y (2011) Natural variation of

drought response in Brachypodium distachyon. Physiologia

Plantarum 141(1): 19–29.

McNeil SD, Nuccio ML and Hanson AD (1999) Betaines and

related osmoprotectants. Targets for metabolic engineering of

stress resistance. Plant Physiology 120(4): 945–950.

Ogasawara Y, Ishizaki K, Kohchi T andKodamaY (2013) Cold-

induced organelle relocation in the liverwort Marchantia poly-

morpha L. Plant, Cell & Environment 36(8): 1520–1528.

Ouellet F (2002) Out of the cold: unveiling the elements required

for low temperature induction of gene expression in plants.

In Vitro Cellular and Developmental Biology – Plant 38(5):

396–403.

Pavangadkar K, Thomashow MF and Triezenberg SJ (2010)

Histone dynamics and roles of histone acetyltransferases during

cold-induced gene regulation in Arabidopsis. Plant Molecular

Biology 74(1–2): 183–200.

Peraldi A, Beccari G, Steed A and Nicholson P (2011) Brachy-

podium distachyon: a new pathosystem to study Fusarium head

blight and other Fusarium diseases of wheat. BMC Plant Biol-

ogy 11: 100.

Rogers K and Chen X (2013) Biogenesis, turnover, and mode of

action of plant microRNAs. Plant Cell 25(7): 2383–2399.

Schwartz CJ, Doyle MR, Manzaneda AJ et al. (2010) Natural

variation of flowering time and vernalization responsiveness in

Brachypodium distachyon. BioEnergy Research 3(1): 38–46.

Steimer A, Schob H and Grossniklaus U (2004) Epigenetic con-

trol of plant development: new layers of complexity. Current

Opinion in Plant Biology 7(1): 11–19.

Steponkus PL (1984) Role of the plasma membrane in freezing

injury and cold acclimation.Annual Review of Plant Physiology

35: 543–584.

Stockinger EJ, Mao Y, Regier MK, Triezenberg SJ and Thoma-

show MF (2001) Transcriptional adaptor and histone acetyl-

transferase proteins in Arabidopsis and their interactions with

CBF1, a transcriptional activator involved in cold-regulated

gene expression. Nucleic Acids Research 29(7): 1524–1533.

Sunkar R, ChinnusamyV, Zhu J and Zhu JK (2007) Small RNAs

as big players in plant abiotic stress responses and nutrient

deprivation. Trends in Plant Science 12(7): 301–309.

Suzuki N, Koussevitzky S, Mittler R and Miller G (2012) ROS

and redox signalling in the response of plants to abiotic stress.

Plant, Cell & Environment 35(2): 259–270.

Tang Z, Zhang L, Xu C et al. (2012) Uncovering small RNA-

mediated responses to cold stress in a wheat thermosensitive

genic male-sterile line by deep sequencing. Plant Physiology

159(2): 721–738.

The International Brachypodium Initiative (2010) Genome

sequencing and analysis of the model grass Brachypodium dis-

tachyon. Nature 463(7282): 763–768.

Thomas GH and Elgin SC (1988) Protein/DNA architecture of

the DNase I hypersensitive region of the Drosophila hsp26

promoter. EMBO Journal 7(7): 2191–2201.

ThomashowMF (1999) Plant cold acclimation: freezing tolerance

genes and regulatory mechanisms. Annual Review of Plant

Physiology and Plant Molecular Biology 50: 571–599.

Tremblay K, Ouellet F, Fournier J, Danyluk J and Sarhan F

(2005)Molecular characterization and origin of novel bipartite

cold-regulated ice recrystallization inhibition proteins from

cereals. Plant & Cell Physiology 46(6): 884–891.



Trevaskis B, Tadege M, Hemming MN et al. (2007) Short vege-

tative phase-like MADS-box genes inhibit floral meristem

identity in barley. Plant Physiology 143(1): 225–235.

Uemura M, Tominaga Y, Nakagawara C et al. (2006) Responses

of the plasma membrane to low temperatures. Physiologia

Plantarum 126(1): 81–89.

Vlachonasios KE, Thomashow MF and Triezenberg SJ (2003)

Disruption mutations of ADA2b and GCN5 transcriptional

adaptor genes dramatically affect Arabidopsis growth, devel-

opment, and gene expression. Plant Cell 15(3): 626–638.

Vogel JP, Tuna M, Budak H et al. (2009) Development of SSR

markers and analysis of diversity in Turkish populations of

Brachypodium distachyon. BMC Plant Biology 9: 88.

Vogel JT, Zarka DG, Van Buskirk HA, Fowler SG and Tho-

mashow MF (2005) Roles of the CBF2 and ZAT12 transcrip-

tion factors in configuring the low temperature transcriptome

of Arabidopsis. Plant Journal 41(2): 195–211.

Zhang J, Xu Y, Huan Q and Chong K (2009) Deep sequencing of

Brachypodium small RNAs at the global genome level identifies

microRNAs involved in cold stress response. BMC Genomics

10: 449.

Zhang X, Xia J, Lii YE et al. (2012) Genome-wide analysis of

plant nat-siRNAs reveals insights into their distribution, bio-

genesis and function. Genome Biology 13(3): R20.

Zhou X, Wang G, Sutoh K, Zhu JK and Zhang W (2008) Iden-

tification of cold-inducible microRNAs in plants by

transcriptomeanalysis.Biochimica etBiophysicaActa 1779(11):

780–788.

Zhu J, Jeong JC, Zhu Y et al. (2008) Involvement of Arabidopsis

HOS15 in histone deacetylation and cold tolerance. Proceed-

ings of the National Academy of Sciences of the USA 105(12):

4945–4950.

Further Reading

He G, Elling AA and Deng XW (2011) The epigenome and plant

development. Annual Review of Plant Biology 62: 411–435.

Oono Y, Seki M, Satou M et al. (2006) Monitoring expression

profiles of Arabidopsis genes during cold acclimation and

deacclimation using DNA microarrays. Functional and Inte-

grative Genomics 6(3): 212–234.

Sarhan F andDanyluk J (1998) Engineering cold-tolerant crops–

Throwing the master switch. Trends in Plant Science 3(8): 289–

290.

Vigeland MD, Spannagl M, Asp T et al. (2013) Evidence for

adaptive evolution of low-temperature stress response genes in

a Pooideae grass ancestor.New Phytologist 199(4): 1060–1068.

Zilberman D, Coleman-Derr D, Ballinger T and Henikoff S

(2008) Histone H2A.Z and DNA methylation are mutually

antagonistic chromatin marks. Nature 456(7218): 125–129.



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