The molecular biology of plant acclimation to low temperature

Journal of Experimental Botany, Vol. 47, No. 296, pp. 291-305, March 1996Journal ofExperimentalBotany

REVIEW ARTICLE

The molecular biology of plant acclimation to lowtemperature

Monica A. Hughes1 and M. Alison Dunn x

Department of Biochemistry and Genetics, The University of Newcastle upon Tyne, Newcastle upon TyneNE24HH, UK

Received 6 June 1995; Accepted 2 November 1995

ABSTRACT

In many temperate plant species, a period of exposureto a low positive temperature will acclimate the plantsto withstand a subsequent freezing stress. A numberof plant genes, which are up-regulated at steady-statemRNA levels by an acclimation treatment, have beenisolated from both monocotyledon and dicotyledonspecies. Most of these genes are also responsive to adrought treatment and/or abscisic acid. The acclima-tion of plants to freezing stress is a complex processand although genetic studies in some species haveidentified genes with a major effect, in general, theinheritance of frost tolerance is multigenic. In view ofthis, it is not surprising that a range of different geneshave been cloned. The precise function of the proteinsencoded by these genes is unknown. However, ana-lysis of predicted protein products and studies ofrecombinant proteins, together with detailed expres-sion studies, are beginning to provide informationabout some of the genes. Both transcriptional andpost-transcriptional controls have been shown to beinvolved in the expression of these genes. Althoughstudies of RNA stabilizing systems are still in theirearly stages, a number of low temperature responsivepromoters have been studied using reporter geneconstructs. Other approaches to the molecularanalysis of cold acclimation include the isolation ofnon-acclimating mutants and the production oftransgenic plants.

Key words: Abscisic acid, cold acclimation, drought, frosttolerance, low temperature.

Introduction

Acclimation for frost tolerance

In many overwintering temperate plant species a periodof low positive temperature will increase tolerance to asubsequent subzero (freezing) temperature. This processis known as cold or frost acclimation and is acknowledgedto be complex, involving a number of biochemical andphysiological changes. Cellular and metabolic changesthat occur during cold acclimation include increased levelsof sugars, soluble proteins, proline, and organic acids aswell as the appearance of new isoforms of proteins andaltered lipid membrane composition (Hughes and Dunn,1990). There is an increasing body of evidence to showthat many of these biochemical and physiological changesare regulated by low temperature through changes ingene expression and in recent years a number of low-temperature-responsive (LTR) genes have been clonedfrom a range of both dicotyledon and monocotyledonspecies.

The ability to acclimate to frost tolerance is undergenetic control and a low temperature of 6 °C to 2 °C,which will cold acclimate genetically competent plants,such as winter cereals, will damage a chill-sensitive species,such as maize. However, not all chill-tolerant plants areable to acclimate for frost tolerance and many springcereal cultivars can be classified in this group. In additionto acclimation for frost tolerance, a low positive temper-ature treatment may also acclimate chill-tolerant plantsfor growth at low temperatures (Hughes and Pearce,1988). In species and cultivars which will cold acclimate,the low temperature treatment may also alter the growthhabit and vernalize the plants. Further, there is often aninteraction between low temperature and other environ-mental factors, particularly daylength and water status(Pan et cil., 1994).

1 To whom correspondence should be addressed. Fax +44 191 222 7424.

© Oxford University Press 1996

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292 Hughes et al.

Genetic analysis of cold acclimation

The genetic analysis of cold acclimation has been carriedout primarily in cereals. Winter hardiness of a cereal cropis composed of a number of interacting components, frosttolerance, vernalization requirement and photoperiodsensitivity, each of which is complex. In the diploid cerealbarley it has been suggested that a change in the growthhabit caused by the vernalization response can contributedirectly to winter hardiness of the crop, however, Dollet al. (1989) were able to separate vernalization require-ment and good frost tolerance in a number of doubledhaploid lines produced by crossing a winter, frost hardycultivar (Vogelsanger Gold) with four spring, frost-sensitive cultivars (Tron, Tystofte Prentice, Clipper, andAlf). Takahashi and Yasuda (1970) concluded that therewere three major genes for vernalization requirementlocated on barley chromosomes 4, 7 and 5. More recentlyHackett el al. (1992) mapped a quantitative trait locus(QTL) for vernalization on barley chromosome 4although this locus may strictly be a heading (flowering)date locus (Pan et al., 1994). A 74-point linkage analysisof a spring x winter barley (Morex x Dicktoo) cross byPan et al. (1994) has identified a significant QTL forwinter survival on chromosome 7.

The homeologous wheat chromosome for barley chro-mosome 7 is chromosome 5. In hexaploid wheat fivevernalization loci have been identified. Vrn 1, Vrn 4 andVrn 3 are located on chromosome 5A, 5B and 5D,respectively (Xin et al., 1988). Vrn 2 is located onchromosome 2B (homeologous barley chromosome 2),and Vrn 5 is on chromosome 7B (homeologous barleychromosome 1) (Law, 1966). In addition, the Sp\ genefor vernalization has been mapped to the rye chromosome5, which is also homeologous to wheat chromosome 5(Plaschke et al., 1993). Of the chromosomes identified ascarrying genes involved in frost tolerance in wheat, thosemost frequently implicated are chromosomes 5A, 7A, 4D,and 5D (Sutka and Snape, 1989). Sutka and Snape (1989)have reported a locus having a major effect on frosttolerance (Fr\) on chromosome 5A which is closely linkedto the recessive vrn 1 allele (Sutka et al., 1996) andRoberts (1990) identified two loci on wheat chromosome5A which are involved in frost tolerance.

These genetic studies only reveal loci which are involvedin the response of cereals to low temperatures, where theparents used in the cross contain different alleles whichsegregate in the progeny of the F, hybrid or alter thegenotype of cytological substitution or addition lines. Thepower of this analysis to dissect the processes of frostacclimation depends, therefore, on the amount of vari-ation available within the crop (species).

Mutational and transgenic studies of frost tolerance

The plasma membrane is considered a primary site ofinjury during freezing (Lyons et al., 1979) and membrane

lipid composition is widely considered to be important intolerance to low temperatures. Freezing of plant tissuesresults in the formation and growth of extracellular icecrystals, this inflicts a dehydrative stress on cells. Injurydue to freezing can be caused by lesions in the plasmamembrane that result in loss of osmotic responsivenessduring subsequent thawing (Steponkus, 1984). Plants thathave been grown at low temperatures show an increasein the level of lipid unsaturation (Harwood et al., 1994),but changes in lipid composition and the ratio of lipid toprotein in membranes may also change (Harwood et al.,1994; Palta et al., 1993). Roughan (1985) has surveyedthe fatty acid composition of phosphatidylglycerol in 74plant species, to show a broad correlation between chillsensitivity and the percentage of saturated plus trans-mono-unsaturated phosphatidylglycerol. More directevidence that changes in phospholipid content affect thecryo-behaviour of plasma membranes has been producedby Steponkus et al. (1988) using a liposome-protoplastfusion technique. Liposomes of different molecular speciesof phosphatidylcholine were fused with protoplasts fromnon-acclimated rye seedlings to selectively enrich theplasma membrane. The modified protoplasts showed thatincreased levels of mono- and di-unsaturated species ofphosphatidylcholine mimic the behaviour of protoplastsfrom acclimated plants and suffer less expansion-inducedlysis during thawing.

The overwintering annual species Arabidopsis thalianais able to acclimate for frost tolerance (Lin et al., 1990)and shows a low temperature vernalization response(Chandler and Dean, 1994). Figure 1 shows a summaryof the mutations of Arabidopsis which affect lipid biosyn-thesis and which have been evaluated for their effect oncold tolerance. The fad 2 mutation results in plants witha deficiency in the production of polyunsaturated (low-melting point) fatty acids and these plants are killed bylong exposure to low (non-freezing) temperatures (Miguelet al., 1993). Similarly, fad 5 and fad 6 mutants aredeficient in unsaturated lipids, but these mutations affectlipids within the chloroplasts and the mutant plants showdefects in normal chloroplast development at low tem-peratures (Hugly and Sommerville, 1992). All of thesemutant Arabidopsis are chill-sensitive and can, therefore,no longer acclimate for frost tolerance. Tht fab 1 muta-tion, results in an increased level of 16:0 (saturated) fattyacids compared with wild type. Recently, Wu and Browse(1995) have shown that although fab 1 Arabidopsis mut-ants contain a higher level of saturated (high-melting-point) fatty acids in phosphatidylglycerol compared withmany chill-sensitive species, these mutants are not chill-sensitive. This observation is not in agreement with theprevious results.

An outline of studies which have manipulated levels ofunsaturated fatty acids in plants by transformation isshown in Fig. 2. Murata et al. (1992) transformed tobacco


Gene Biochemical function

Molecular biology of plant acclimation 293

Chffl sensitivity

fadi

fads

fade

111:1

I11:1

I18:1

I18:1

I18:1

I18:0

I16:1

I -16:0

—> r~18:2

> I "18:1

—> r~18:2

—> r~18:2

I18:2

I16:1

I16:2

I16:0

(ER)

(plastid)

(frfastid)

(plastid)

fab\ 16:0 ACP ^ 18:0 ACP

Fig. 1. Mutations of Arabidopsis thaliana which affect lipid biosynthesis and have been evaluated for their effect on chill tolerance.

chilling sensitive

lobacco

intermediate

Transgenictobacco

chilling sensitive

SiArabidopsis

W chiding resistant

Arabidopsis

chilling sensitive

E.coli

prokaryote

\

transferase3

acyt-ACP : glycerol-3-phosphate acyttransferase 1 cannot dlscrimlnata between saturated and cto-unsaturated fatty addsacyt-ACP: glycerol-3-phospriate acyttiaiisfaiMsa 2 has preferential activity for cfe-unsaturatad fatty addsacyt-ACP : glycaroKH>nosphate acyttransferase 3 cannot discriminate between 16:0-ACP and 18:OACPa>-3 fatty add desaturase (tad 7) converts 18 J and 16:2 to 16:3 and 16:3 respectively.

Fig. 2. Manipulation of chill-sensitivity in Nicotiana labacum and Arabidopsis thalianu by transformation.

(which was considered to have an intermediate level ofchill-sensitivity) with acyl-ACP: glycerol-3-phosphateacyltransferase from either chill-sensitive squash orchill-resistant Arabidopsis. This resulted in higher levelsof high-melting-point (saturated) phosphatidylglycerol in

the squash enzyme transformant and lower levels in theArabidopsis enzyme transformant. In addition, the squashenzyme transgenic tobacco plants had increased chill-sensitivity whilst the Arabidopsis enzyme transgenic plantswere more chill-resistant.


294 Hughes et al.

Two other groups have manipulated levels of unsatur-ated lipids by transformation and produced a change inthe chill-sensitivity of the resulting plants. Wolter et al.(1992) transformed Arabidopsis with an E. coli acyl-ACP:glycerol-3-phosphate acyltransferase which can notdiscriminate between 16:0-ACP and 18:0-ACP. Theresulting transgenic plants contain more saturated phos-phatidylcholine and are chill-sensitive. More recently,Kodama et al. (1994) produced transgenic tobacco thatcontain an w-3 fatty acid desaturase gene (fad 7) fromArabidopsis, these plants contain increased levels of poly-unsaturated lipids and an increase in resistance to chillingtemperatures.

These studies, where a single gene is manipulated,provide powerful evidence for the involvement of polyun-saturated lipids in the resistance of membranes to lowpositive temperatures and although this may not bedirectly involved in frost acclimation, clearly plants mustbe chill-tolerant in order to acclimate at the low (chilling)temperature. The anomalous behaviour with respect tochill-sensitivity, of the fab 1 mutants (Wu and Browse,1995) highlights the complexity of low temperatureresponses in plants. Wu and Browse (1995) comment onthe possible pleiotropic effects of some of the transgenesand the difficulty of interpreting some of the single genemodifications for a multigenic character, in plants withvery different genetic backgrounds.

Low-temperature-responsive (LTR) genes

Overview of LTR genes

Details of most of the LTR genes which have been studiedto date are shown in Table 1. Twenty of the gene/genefamilies come from dicotyledon species and 11 frommonocotyledons. Although this list contains a wide rangeof different genes there are some general points which aredemonstrated by the compilation. Most of the genes wereisolated from cDNA libraries using a differential screeningprocedure where normal growth temperatures and a lowpositive (acclimating) temperature were used in the gen-eration of different pools of mRNA. This technniqueisolates genes which are up-regulated by low temperatureat steady-state mRNA levels and where the mRNAspecies is present at relatively high levels in the mRNApopulation. RC\ and RC2 were, however, isolated by atechnique designed to identify rare transcripts (Jarilloet al., 1994).

A number of the genes have been isolated from thesame species independently by different groups (e.g. cor6.6 (kin 1); Hajela et al., 1990; Kurkela and Franck,1990). In addition, cognates of some genes have beenindependently isolated from several species (e.g. /// 30from Arabidopsis (Welin et al., 1994); cap 85 from spinach(Neven et al., 1993); cor 39 from wheat (Guo et al..

1992)). Ten gene/gene families have been isolated fromArabidopsis and 6 gene/gene families from barley. Thisnumber of LTR genes in one plant species is consistentwith the acknowledged complexity of the frost acclimationprocess. It is considered unlikely that this is an exhaustivelist of LTR genes since the technique employed to isolatethem will not identify genes expressed at low levels orgenes transiently expressed during the acclimation pro-cess. The nature of the material used as a source ofmRNA is also a factor in the isolation of these genes.Thus one possible explanation for the fact that, with theexception of Ccr 1, none of the Arabidopsis LTR geneshave been cloned in barley, may be that different materialwas used as a source of the cDNA library. Five of thesix barley genes were isolated from a cDNA library madefrom shoot meristems, whereas the Arabidopsis librarieswere made from mRNA extracted from whole plants,largely made up of mature leaves.

Role of gene families

Many of the genes which have been the subject ofextensive studies are members of small multigene families.In Arabidopsis cor 6.6 (kin 1) and kin 2 are linked intandem, they are expressed at different levels in responseto low temperature and they also differ in their responseto drought (Kurkela and Borg-Franck, 1992). Similarly,cor 15A and cor 15B from Arabidopsis are linked intandem and, although cor 15A is responsive to both lowtemperature and drought, cor 15B is only responsive tolow temperature (Lin and Thomashow, 1992; Wilhelmand Thomashow, 1993).

The complexity of the relationship between genes inLTR multigene families is illustrated in barley. Fourmembers of the bit 4 gene family have been cloned (Dunnet al., 1991; White AJ et al., 1994). These LTR genes arealso responsive to drought and ABA, but the relativeresponse to these three factors varies between the indi-vidual genes. The gene bit 4.9 is strongly induced by lowtemperature and only slightly induced by drought,whereas bit 4.1 is equally responsive to both treatments(Fig. 3). There is also a temporal control of expressionof bit 4.2, which is only expressed in seedlings (White AJet al., 1994).

There are only two members of the bit 101 familypresent in the barley genome. In this pair of genes bothare responsive to low temperature and the cDNAsequences are over 70% homologous. Because of the levelof homology between the two genes it was impossible tobe confident of the use of gene specific probes and aquantitative reverse transcriptase PCR technique wasused to measure relative levels of expression. Figure 4shows the result of using this technique to measure theexpression of bit 101.2 in low temperature-treated shootmeristems. A dilution series of cloned genomic DNA,


Table 1. Low temperature responsive genes

DICOTYLEDONS

Species Gene Other membersof gene family

Cognates inother species

Class of proteinsor gene

Other stresses Reference

Arabidopsis lhaliana

Brassica napus(oil seed rape)

Medicago salivasubspp falcata(alalfa)

Spinacia oleracea(spinach)Solatium commersonii

MONOCOTYLEDONS

Hordeum vulgare(barley)

Secale cereale (rye)Triticum aestivum(wheat)

for 6.6 {kin 1)

cor 78

cor 15A

for 47In 30Ili 140rab 18Ccr 1RCI 1AdhBN 115fijV 28BnC 24Acas 15cas 17Msaci AHKWCICcap 85ER-HSC 70A 13

bit 14

W l 4

bh 1016/f 63WM 1bit 801r/( 1412Wcs 120

Wcs 19for 39Wf or 410

A-m 2

In 78, /f; 65rd 29A, rrf 29B

for 15B

//; 45---Cfr 2RCI 2-BN 19, BN 26-BnC 24B-COJ 18SAY 2075----

6// 14.2; bit 14.1A 0866// 4.2; 6// 4.6; bit

bit 101.2--_rlt 1421Wcs 200, Wcs 66

_-

fl/i 28

-

fin 115

-cap 85, for 39

-bit 801--for 15Afor 6.6bbc 1----Iti 30, for 39--

rlt 1412; rlt 1421

-

ESI 2,--Ccr 1bit 14_

In 30, fa/> 85-

AFP

NK

NK

D-ll LEAD-ll LEANKD-ll LEARNABP14-3-3ADHNKAFPNKNKD-ll LEANKproline richD-ll LEAHSPOsmotin

NK

LTP

NKEF-laD-7 LEARNABPNKD-ll LEA

NKD-ll LEAD-ll LEA

Drought, ABA

DroughtABA (salt)

Drought (ABA)

Drought, ABADrought, ABADrought, ABADrought, ABA(Drought)_Drought, hypoxia-NK-_-Drought, ABA-Drought-Drought, ABA

_

Drought, ABA

_

NKABAABA--

-Drought, ABADrought, ABA

Hajela el al. (1990); Kurkela and Franck(1990); Kurkela and Borg-Franck (1992)Horvath et al. (1993); Nordin el al(1993); Yamaguchi-Shinozaki andShinozaki(1994)Lin and Thomashow (1992); Wilhelm andThomashow (1993)Lin et al. (1990); Welin el al. (1994)Welin et al. (1994)Nordin el al. (1991)Lang and Palva (1992)Carpentered al (1994)Jarillo et al. (1994)Dolferus et al. (1994)Weretilnyk el al. (1993)Orr et al (1992)SSez-Vasquez et al. (1993)Monroy et al. (1993)Wolfraim el al. (1993 a, b)Labergeera/. (1993); Luo el al. (1991)Castonguay el al (1994)Neven et al. (1993)Anderson et al. (1994)Zhu elal. (1993)

Dunn el al. (1990);Cativelli and Bartels (1990)Dunn el al. (1991); White et al. (1994)

Goddard et al (1993)Dunn et al. (1993)Sutton et al. (1992)Dunn et al. (1996)Zhang et at. (1992)Houde et al. (1992); Ouellet et al. (1993);Chauvin et al. (1994)Chauvin et al. (1993)Guo et al. (1992)Danyluk el al. (1994)

s2.o

12"

o

I0)oo

Key to abbreviations: AFP = antifreeze proteins; ABA = abscisic acid; LEA = late embyogenesis abundant proteins; RNABP = RNA binding protein; NK=not known; bbc 1 —breast basicconserved gene; HSP»heat shock protein; ADH = alcohol dehydrogenase; 14-3-3 kinase regulatory protein; LTP = hpid transfer protein; EF-1 a°= elongation factor-la; ESI 3 = salt stressinduced gene from wheatgrass; - = none; ( ) bracket indicates stress response of other members of gene family.

I"6'


296 Hughes et al.

WM.1 gWM.B MM.9

D Low Temperature E3 Drought

Fig. 3. Comparative Northern blot analysis of the relative abundanceof bit 4.1, bit 4.6 and bit 4.9 transcripts in barley (cv. Igri) shootmeristems. Histogram of integral of densitometric peaks. Relativeresponse to drought expressed as a percentage of induction by lowtemperature (6°C day/2°C night) (White AJ et al., 1994).

ng/ulplasmid QQQ6 R

P = plasmid PCR product

R = RT PCR product

Fig. 4. Quantitative reverse transcriptase (RT) PCR: expression of thebarley gene bit 101.2 in shoot meristems given a 7 day treatment at6°C day/2 °C night (10 h day). The PCR product from cloned genomicDNA is 450 bp and includes the intron, the RT PCR product fromidentical aliquots of a mRNA extract is 378 bp. The dilution series ofcloned genomic DNA used was 4.0 ng j j ' 1 to 0.006 ng ^ l 1

which contains an intron, is added to PCR of a constantlevel of cDNA produced from idential aliquots of anmRNA extract. The primers anneal to sites either side ofthe intron and, therefore, produce different sized productsfrom the two templates. The point of equivalence ofquantity of the two PCR products is a measure of thelevel of cDNA (mRNA) in the extract. This reaction isrepeated using mRNA from control temperature plantsand with genomic DNA and specific primers for bit 101.This study has shown that bit 101 is expressed at 10 timesthe level of bit 101.2 at both control and low temperature.Three members of the bit 14 gene family have also been

cloned from barley and these genes show a pattern ofregulation similar to that found in the cognate gene familyin rye (Zhang et al., 1993). That is, all of the genes areresponsive to low temperature and not drought or ABA,but the organ distribution of gene expression is differentfor each gene (Phillips and Hughes, unpublished).

Smith (1990) suggested that phenotypic plasticity ofplants in response to different environmental factors maybe due to the differential expression of different membersof multigene families. This can be extended to the cellularor metabolic level where different environmentalconditions require modification of cell structure and/ormetabolism in order to maintain vital processes. Thesemodifications may require different protein isoformsunder different environments. This hypothesis providesan explanation for the pattern seen in the bit 4 genefamily. However, the production of different bit 14 iso-forms in different parts of the plant is consistent with thesuggestion that the modification of cells in different tissuesmust be modulated during the low temperature acclima-tion process.

Response to other stresses

Seventeen of the 30 LTR gene/gene families tested arealso responsive to drought and/or abscisic acid (Table 1).In addition, two of the genes have cognates which areresponsive to salt stress. At least 3 of the bit 4 gene familyare also induced by pathogen infection (Table 2; Molinaand Garcia-Olmedo, 1993). Because, during freezing, icecrystals form outside the cells, both freezing and droughtare considered dehydrative stresses (Steponkus, 1984). Itis reasonable, therefore, to suggest that similar modifica-tions to the cell may be required for tolerance to bothstresses and the production of common gene productsmay therefore be induced. A rapid transfer to a lowpositive temperature will cause a reduction in root wateruptake and a transient 'locking-open' of stomata causingtransient loss of plant turgor (Pardossi et al., 1994).However, the low positive temperature treatment whichinduces the expression of the LTR genes is not primarilya dehydrative stress and the extent to which differentsignal transduction pathways are involved in controllingthe expression of genes by drought and low positivetemperature remains to be determined.

Function of gene products

Including the D-11 (dehydrin) class of late embryogenesisabundant (LEA) related genes, there is no known bio-chemical function for 26 of the 31 LTR gene/gene familieslisted in Table 1. A number of studies have investigatedboth freezing tolerance and levels of gene expression inthe same genotype under a range of acclimating treat-



Table 2. Barley lipid transfer protein genes

Gene

Itp 1Itp 3W/4.1 (Itp 2)bit 4.2 (Itp 4)bit 4.6bit 4.9

Chromosome

753333

Presenceof introns

yyNK———

3 amino aciddeletions atR82

—yyyy

Response

NK—LTLTLTLT

NKNKDroughtNKDroughtDrought

NK,PathogenPathogenPathogenNKPathogen

NKABAABAABANKABA

Tissuedistribution

Aleurone layerNKLeafColeoptileNKShoot meristem

— = None; NK = not known

ments. Monroy et al. (1993a) showed that changes infreezing tolerance of alfalfa cv. Apica are paralleled bychanges in the transcript levels of cas 15. In barley, thesteady-state mRNA levels of three LTR genes were meas-ured at a range of temperatures during acclimation anddeacclimation. In this study the level of frost tolerance(LTJ0 in a regrowth test) was shown to correlate with thelevel of gene expression (Pearce et al., 1996).

The relationship between levels of gene expression andthe ability of different genotypes to acclimate for frosttolerance has also been investigated. Danyluk et al. (1994)have shown that Wcor 410 is only expressed in freezing-tolerant gramineae. Houde et al. (1992) have shown thatthe accumulation kinetics of Wcs 120 mRNA duringacclimation is positively correlated with the capacity ofeach wheat genotype to develop frost tolerance. Similarly,Zhang et al. (1992) showed that the low temperatureinduced steady-state mRNA levels of two rye LTR genes(rlt 1412, rlt 1421) were lower in the relatively frost-sensitive rye cultivar, Rhayader, compared with the frost-hardy cultivar, Puma. These comparative studies supportthe proposal that these LTR genes have a function infrost acclimation.

Low temperature isoforms

Biochemical studies have suggested that growth at lowtemperature will result in the production of low temper-ature isoforms of enzymes involved in 'housekeeping'functions. With the exception of the barley protein syn-thesis elongation factor-la (EF-la, Dunn et al., 1993),this type of gene is not present in Table 1. The lack ofthese genes may be due to the cloning strategy commonlyused. Many of the low-temperature isoforms of proteinscan be expected to have similar structures and, con-sequently, the coding regions of the mRNA will be highlyhomologous and cross hybridize. This means that suchclones would not be detected easily in a differential screen.The barley EF-la is a highly conserved protein, it is amember of a multigene family in the barley genome andthe low temperature induction of bit 63 can only be seen

in Northern blot analysis if the 3' untranslated sequenceof the mRNA is used as a probe.

Cryo-protection

A number (8) of the LTR genes in Table 1 are predictedto encode proteins with the characteristics of the D-l 1 ordehydrin class of LEA proteins. These proteins accumu-late during the late stages of seed development and arepredicted to have a role in dehydration of the seed. Thedehydrins are characterized by a repeated consensus 15amino acid sequence near the C terminus of the proteinand, in addition, many have a tract of serine residuesupstream of these domains (Close et al., 1993). Theproteins are very hydrophilic and are 'boiling stable'.They were originally thought to act as osmoprotectantsalthough it has recently been suggested that they mayhave a role in nuclear protein transport (Close et al.,1993).

The chloroplast targeted protein product of cor 15 A inArabidopsis is also hydrophilic and 'boiling stable' (Linand Thomashow, 1992) and this protein has been shownto have cryoprotective activity in vitro against the freeze-labile enzyme L-lactate dehydrogenase. It is difficult toextrapolate from this in vitro assay for cryoprotectiveactivity, to the in vivo function, but the common occur-rence of 'boiling stable' hydrophilic proteins amongst theLTR genes makes cryoprotection an attractive role forthem. Two other genes (cor 6.6 from Arabidopsis and BN28 from oil seed rape) have homology to antifreezeproteins, thus cor 6.6 {kin 1) bears some compositionalsimilarity to the fish alanine-rich antifreeze proteins(Kurkela and Franck, 1990). These clones support thesuggestion that a significant proportion of the LTR genescloned have a cryoprotective function. Hincha et al.(1990) have used an in vitro test for thylakoid membranecryoprotection against mechanical freeze-thaw damage,to study protein extracts from leaves of cabbage andspinach. Their results show that proteins with significantcryoprotective activity are only isolated from frost-


298 Hughes et al.

acclimated plants. It will be interesting to see the structureof these proteins when they have been purified.

Membrane/lipid modifications

It is perhaps surprising, given the importance of mem-brane lipid composition in chill-tolerance and frost accli-mation (outlined in 'Mutational and transgenic studies offrost tolerance'), that none of the genes in Table 1 has adirect role in the synthesis of these compounds. The onlygene family for which a lipid related function is proposedis the bit 4 gene family in barley, which is predicted toencode a group of non-specific lipid transfer proteins(White AJ et al., 1994).

A summary of the lipid transfer protein (LTP) geneswhich have been cloned in barley is shown in Table 2.This table, which includes the LTP gene (Up 1) expressedin aleurone cells during germination (Mundy and Rogers,1986), a pathogen induced LTP gene (Up 3) and twocognates {Up 2, Up 4) of the bit 4 gene family isolatedfrom the spring cultivar, Bomi (Molina and Garcia-Olmedo, 1993) as well as the winter barley bit 4 LTPgenes (White AJ et al., 1994), shows the complexity oflipid transfer proteins produced by a single species. Onthe basis of gene structure these genes may be dividedinto two groups Up 1 and Up 3 being distinguished fromthe bit 4 genes by their chromosomal location, the pres-ence of an intron in the gene and an insertion of ninebases in the mRNA coding sequence, which leads to theaddition of 3 amino acids at residue position 85 in thepolypeptide. Molina and Garcia-Olmedo (1993) haveshown that Up 3, bit 4.1 (Up 2) and bit 4.2 (Up 4) areresponsive to pathogen stress, but not low temperature,in cv. Bomi. The low temperature, drought and ABAresponse of the bit 4 gene family (White AJ et al., 1994,Hughes et al., 1992) and the pathogen response of bit 4.9(Hughes and O'Hara, unpublished) have been measuredin the winter cv. Igri. In common with other plant LTPs,none of these genes is expressed in roots and they showsome tissue specificity. LTP have been defined by theirability to transfer lipids between membranes (typicallymitochondria and liposomes) in vitro, however, theirfunction in vivo has been questioned. In barley bit 4.9 hasrecently been shown to be expressed in epidermal cells ofthe leaf sheath bases (Hughes et al., unpublished). All ofthe bit 4 gene family possess a consensus N-terminalsignal sequence for extracellular transport (White AJet al., 1994) and this may not be consistent with a lipidtransfer function related to changes in membrane lipidsduring frost-acclimation. Recently, an extracellular, wax-associated LTP has been isolated from broccoli (Brassicaoleracea) leaves, this is a member of a LTP gene familywhich is expressed in leaf epidermal cells (Pyee et al.,1994; Pyee and Kolattukudy, 1995). These results suggest

that the LTPs may be involved in wax synthesis and/orsecretion. A role in leaf surface biochemistry is consistentwith the response of barley LTPs to a wide range offactors and must be investigated as an alternative to thelipid transfer function.

Low temperature control of gene expression

Signal transduction

Abscisic acid (ABA) is implicated in the plant responseto low-temperature by a number of observations: (1) Anacclimating low-temperature treatment (4CC/2°C) ofArabidopsis lhaliana results in a transient, 3-fold increasein endogenous ABA levels (Lang et al., 1994). Thisincrease is seen in both axenically grown and soil-grownplants and has also been observed in other species (Lalkand Ddrfliing, 1985). (2) Application of ABA can inducean increase in freezing tolerance in Arabidopsis plantsgrown at a control temperature (22°C) (MSntylS et al.,1995). (3) ABA-null mutants of Arabidopsis (aba-\) areimpaired in their ability to cold acclimate with low-temperature treatment (MantylS et al., 1995, Heino et al.,1990). (4) Application of ABA to the ABA-insensitivemutant (abi-l) does not induce cold acclimation (Mantyla'et al., 1995). However, several studies have suggestedthat there is more than one signal pathway for low-temperature responses (Nordin et al., 1991; Gilmour andThomashow, 1991, Jarillo et al., 1993) and this is illus-trated by the ability of abi-\ mutants to cold acclimateto some extent. Table 3 shows a schematic representationof the response of six Arabidopsis genes to low temper-ature, drought and ABA in 'wild type' (Lansberg erectaecotype) and aba-\ and abi-\ mutants (Mantyla et al.,1995; Welin et al, 1994; Nordin et al, 1991; Jarillo et al.,1993; Gilmour and Thomashow, 1991). All of the genesare responsive to ABA, drought and low-temperaturealthough the relative level of the response to these treat-ments varies between the genes. With the exception ofrab 18 (whose reponse to low-temperature is relativelyweak), all of these genes are up-regulated by low temper-ature in both the aba-] and the abi-\ mutants. However,although the ABA response of these genes is similar towild type in the ABA-null mutant (aba-\), ABA treatmentdoes not induce gene expression in the ABA-insensitivemutant (abi-\). These results indicate that these genesare up-regulated independently by low-temperature andABA.

The abi-\ mutation used in these studies is a semi-dominant mutation and this is consistent with the genehaving a regulatory role. The gene has recently beencloned (Leung et al, 1994; Meyer et al, 1994) and theprotein encoded by this gene is predicted to have aC-terminal domain which is homologous to type 2Cserine/threonine protein phosphatase and an N-terminal



Table 3. Response of Arabidopsis low-temperature-responsive genes in ABA-null (aba-l) and ABA insensitive (abi-1) mutants

Gene

hi 140In 30hi 45hi 78rab 18adhL T ^ ^ . O X )

TreatmentLow temperature

Genotypewt

+ + ++ + +

+ + +++ + +-7.4 X

aba-1

+ + ++ + ++ ++ + +0+ +-3.8 3C

abi-1

+ + ++ + ++ + ++ + +±+ +-5.0 >C

Drought

Genotypewt

+ + ++ ++++ + ++-7.0 X

aba-1

NK+0000-3.0 X

abi-1

+ +

+ +

+±0-3.5 X

ABA

Genotypewt

+ + +

+++ + +

-6.5 X

aba-1

+ +

+ + ++ +++ + +

-7.5 X

abi-1

000+±0-3.0 X

domain with a Ca2+ binding site. Figure 5 shows a modelfor ABA signalling in plants (Bowler and Chua, 1994)which incorporates a plasma membrane-localized ABAreceptor (Gilroy and Jones, 1994) and ABA-mediatedincreases in Ca2+ levels in the cytosol. There is evidencefor the involvement of Ca2+ in both ABA (Gehring et ah,1990) and cold (Knight etal, 1991; Monroy etai, 1993ft)responses and the deduced structure of the abi-l geneproduct suggests that Ca2+ may either activate or repressphosphatase activity leading to a change in the phos-phorylation status of another protein further down the

signal transduction pathway. This, in turn, implicates atleast one kinase in the signal transduction pathway. Thecurrent models for ABI 1 functions do not, however,suggest an explanation for the independent low-temperature and ABA cold acclimation responses. A totalof five genes which have ABA-insensitive mutations havebeen isolated (Rock and Quatrano, 1994) in Arabidopsis.Three of these (abi-3, abi-4, abi-5) are seed specific butmutations of the abi-2 gene are epistatic to abi-\ sug-gesting that abi-\ and abi-2 may lie on the same signalpathway. Gilmour and Thomashow (1991) have shown

aba

drought,:LT

abi 1 (dominant)ABA,

j£ drought,(LT):• m m m-m» m iat m.a:

F? • response

ABRE

2+„>*•

.•£. ,«• j * r; -

ABI-1calcium-dependentphosphatase

\

• response

- ABA (LT)

abi 2

Fig. 5. Model for the involvement of ABA in acclimation for frost resistance. Interrupted arrow (-//-•) shows lack of frost acclimation with thefactors indicated (low temperature, LT; abscisic acid, ABA; drought) in the Arabidopsis mutants aba 1 (abscisic acid null); abi 1 (abscisic acid-insensitive) and abi 2 (abscisic acid-insensitive). Proteins and the ABA receptor are shown as shaded blocks. ABRE, abscisic response element inABA responsive genes.


300 Hughes et al.

that abi-2 mutants of Arabidopsis are able to cold-acclimate with a low temperature treatment. But unlikethe abi-l mutant, ABA treatment of the abi-2 mutantplants leads to induction of the low-temperature-responsive genes tested. Although there is progress in ourunderstanding of the ABA signal transduction pathwaythere is essentially no information about the independentlow-temperature signal pathway.

Of the LTR cloned genes listed in Table 1, the candid-ates for a role in signal transduction are limited. TheArabidopsis gene RCI 1, which was isolated as a raretranscript (Jarillo et al., 1994), has homology to a familyof mammalian proteins, known as 14-3-3 proteins, whichare thought to be involved in the regulation of multi-functional protein kinase activity.

was part of a region shown not to be important in thelow temperature response. The role of the A/GCCGACmotif in low temperature control of transcription clearlyrequires further investigation.

In common with a number of environmentally con-trolled genes several of the putative LTR gene promoterscontain G-Box elements (CACGTG) (Williams et al.,1992). This element is the core motif of a number of ex-acting regulatory sequences including the ABA-respons-ive-element (ABRE). To date, insufficient data exist forthe LTR gene promoters to allow an analysis of whichaspects of the G-Box flanking sequences are importanteither in low temperature regulation per se or in theinteraction of low temperature with other factors suchas ABA.

Transcriptional control of gene expression

The promoter regions of a number of LTR genes havebeen cloned and there are several recent functional studiesof LTR gene promoters including the regulation of BN115 from oil seed rape (White TC et al., 1994), cor 15A(Baker et al., 1994), rd 29A (hi 78) (Nordin et al., 1993;Yamaguchi-Shinozaki and Shinozaki, 1994) and adh(Dolferus el al., 1994) from Arabidopsis. These functionalstudies are based on the ability of promoter constructs todrive the low temperature expression of a reporter geneeither in transgenic plants or in a transient expressionassay using a particle delivery system (PDS). White TCet al. (1994) point out that the core sequence A/GCCGACof an 8-bp TGGCCGAC repeated motif from BN 115 isalso present in those regions of the promoter conferringlow temperature induction of rd 29A (hi 78) and cor 15A.Table 4 also shows that the sequence (ACCGAC) ispresent twice in the putative barley (monocot) promoterof bit 4.6 and the related sequence motif (ACCG/CAC/A)is repeated in the putative promoter of bit 4.9. Nordinet al. (1993) noted that it was also present in the putativeArabidopsis promoter regions of cor 6.6 (kin 1), kin 2 andrab 18. It has been proposed that this core sequence motifis part of a low-temperature-responsive-element (LTRE).However, this motif is not present in the low temperature-responsive promoter of bit 101 which can confer a lowtemperature reponse to the GUS (glucuronidase) reportergene in a transient expression assay using PDS and barleyshoot meristems (Hughes et al., unpublished). Further,analysis of the low temperature response of theArabidopsis adh gene promoter by Dolferus et al. (1994)demonstrates that the anaerobic response element (ARE)is also important in the low temperature response of thisgene. The other element or motif identified as importantin the adJi low temperature response is a G-Box elementCCACGTGG between 218 and -209 from the transcrip-tion start site. The adJi gene does have an A/GCCGAC-like motif (ACCGAT) at -337, but this putative LTRE

Post-transcriptional control of gene expression

Nuclear run-on transcription was used to analyse the lowtemperature regulation of nine barley LTR genes (Dunnet al., 1994) in shoot meristems. This experiment showedthat six of the genes (including bit 4.9, bit 101 and bit63) are primarily transcriptionally regulated whilst threeof the genes (including bit 14 and bit 801) are primarilypost-transcriptionally regulated. The involvement of bothforms of regulation was also reported by Hajela et al.(1990) for four Arabidopsis LTR genes, where one genewas found to be transcriptionally controlled and 3 werefound to be post-transcriptionally controlled. In alfalfa,a combination of the techniques of nuclear run-on tran-scription and transcription inhibition by cordycepinduring deacclimation, has shown that cas 18 is regulatedby low temperature at both the transcriptional and thepost-transcriptional level (Wolfraim et al., 1993). Thedemonstration of post-transcriptional controls in the lowtemperature regulation of gene expression in three speciesindicates that this form of regulation may be of generalimportance in frost acclimation.

A further finding which may be significant for possiblepost-transcriptional control mechanisms, is the isolationof two LTR genes which encode RNA-binding proteins,Ccr 1 and Ccr 2 from Arabidopsis (Carpenter et al., 1994)and bit 801 from barley (Dunn et al., 1996). These genesencode proteins of 16 000-17 000 Mr with two distinctdomains, the amino-terminal domain contains consensusRNP1 and RNP2 motifs, which together with a numberof other highly conserved residues comprise a consensusRNA-binding domain (RRM) of about 80 amino acids.This RRM is found in many RNA binding proteins ofthe nucleus, cytoplasm and cytoplasmic organelles(Dreyfuss et al., 1993). The carboxy-terminal domainconsists of repeating glycine residues interspersed withtyrosine and arginine. The domain structure of bit 801 isshown in Fig. 6.

There have been a number of other reports of stress



Table 4. Low temperature response elements (LTREJ

Species Gene Other members Cognates inof gene family other species

Putative LTRE Experimentalevidence for

Reference

Oilseed rape

Arabidopsis

Barley

BN 115

cor 15A

hi 78

hi 65

bit 4 6

bit 4.9

BN 19, BJV26

cor 15B

In 65, rd 29A

hi 78

bit 4.2, bit 4.9

bit 4 2, bit 4.6

cor 15A

fl/V 115

g«g

gatggttg

catgtgg

teat

tattcaatgg

atgc

atgcagcaagca

GCCGAC

GCCGACGCCGAC

GCCGACACCGAC

ACCGAC

ACCGACGCCGACACCGAC

ACCGAC

ACCGACACCCACACCGAA

gtata

ctgttataca

ctgettact

atcag

atgagaaaataaa

aggt

agttagatatta

LTresponse

•J

J•J

•J

•J

V

yV•j

Droughtresponse

•J (rd 29A)

</ (rd 29A)

White TC et al(1994)

Baker et al.(1994)

Nordin et al.(1993)Yamaguchi andShinozaki (1994)

Nordin et al.(1993)White AJ et al.(1994)

DOMAIN STRUCTURE OF BLT801 PROTEIN

RNPII

1 8 13

RNPI

47 54 151152 161 aa

t(87)

t(126)

glycine rich domain

cyclic AMP-dependent protein kinase (PKA)phosphorylation consensus motif t

RGG Box

Phosphorylatedserine

V

octomer RNPI

hexamer RNPII .

RNA recognition motift Putative proteolytic

site

Fig. 6. Domain structure of the RNA-binding protein encoded by the barley low temperature responsive gene, bit 801

responsive plant genes which encode glycine rich-RNAbinding proteins (GR-RNPs): Zea mays, drought, ABA(Ludevid et al., 1992); Daucus carota, wounding (Sturm,1992); Z. mays, heavy metals (Didierjean et al., 1992);Brassica napus, ABA (Bergeron et al., 1993). Although

this group of genes are all predicted to encode RNAbinding proteins only three have had nucleic acid bindingproperties experimentally verified, namely; MA 16 fromZ. mays (Ludevid et al., 1992); RGP-lb, a GR-RNPwith no reported environmental response from Nicotiana


302 Hughes et al.

silvestris (Hirose et al., 1993) and BLT 801 from barley(Dunn et al., 1996). MA16 and RGP-lb show in vitrobinding preference for poly (U) and poly (G) homori-bopolymers and BLT 801 binds to poly (G), poly (A)and poly (U) homo-ribopolymers, but not poly (C). BLT801 and RGP-lb also bind single-stranded DNA. Thetwo-domain organization of plant GR-RNPs hassimilarity to the Al and A2/B1 groups of heterogeneousnuclear RNA binding proteins (hnRNP) found in animals.These hnRNPs are approximately 34 000 A/r (cf. plantGR-RNPs 17 000 Mr), and are comprised of two domains,an amino-terminal domain containing two RRMs and aglycine rich carboxy-terminal domain of similar composi-tion to plant GR-RNPs. Each domain is approximatelytwice the size of those found in plants (Dreyfus et al.,1993). These animal proteins are located in the nucleusand have been implicated in pre-mRNA splicing includingalternative splicing. The GR-RNPs from Z. mays (Albaet al., 1994) has been localized in the nucleolus and thatof S. alba (Heintzen et al., 1994) to the nucleus, bothwere shown to be most strongly expressed in shootmeristems.

In common with animal hnRNPAl, BLT 801 containsa consensus phosphorylation site for cyclic AMP depend-ent protein kinase (PKA) at the junction between theRNA binding and glycine rich domains. Co-operativeRNA binding and DNA annealing properties, independ-ent of the RRM, have been demonstrated for hnRNPAlin vitro (Cobianchi et al., 1993) with the latter DNAannealing function removed by phosphorylation. In vitrophosphorylation of BLT 801 by PKA has been demon-strated (Dunn et al., 1996) but the biological significanceof this remains to be determined. However, the analogyto the animal heteronuclear proteins is clear and it istempting to speculate that these GR-RNPs represent theplant equivalent involved in alternative splicing of tran-scripts in response to stress or other environmental cues.

Summary

Table 1 documents the considerable activity in the fieldof the molecular biology of plant acclimation to lowtemperature, which has been reported in the past fewyears. As yet, however, there is still no coherent explana-tion of the acclimation response of plants to low temper-ature at a molecular or biochemical level. Progress hasbeen made in the study of low temperature responsivepromoters, some interesting results on post-trans-criptional controls are emerging and there is work in thearea of signal transduction. Perhaps surprisingly, a majorgap in the molecular work is that it has not yielded muchinformation about the function of the low temperatureresponsive genes and this must be a major challenge forthe future.

References

Alba MM, Culianez-Macia FA, Goday A, Freire MA, Nadal B,Pages M. 1994. The maize RNA-binding protein, MA 16, isa nucleolar protein located in the dense fibrillar component.The Plant Journal 6, 825-34.

Anderson JV, Li Q-B, Haskell DW, Guy CL. 1994. Structuralorganization of the spinach endoplasmic reticulum-luminal70 kilodalton heat-shock cognate gene and expression of 70kilodalton heat-shock genes during cold acclimation. PlantPhysiology 104, 1359-70.

Baker SS, Wilhelm KS, Thoraashow MF. 1994. The 5' region ofArubidopsis thaliana cor 15a has m-acting elements thatconfer cold-drought- and ABA-regulated gene expression.Plant Molecular Biology 24, 701-13.

Bergeron D, Beauseigle D, Bellemare G. 1993. Sequence andexpression of a gene encoding a protein with RNA-bindingand glycine-rich domains in Brassica napus. Biochimica etBiophysica Ada 1216, 123-5.

Bowler C, Chua N-H. 1994. Emerging themes in plant signaltransduction. The Plant Cell 6, 1529-41.

Carpenter CD, Krebs JA, Simon AE. 1994. Genes encodingglycine-rich Arabidopsis thaliana proteins with RNA-bindingmotifs are influenced by cold treatment and an endogenouscircadiam rhythm. Plant Physiology 104, 1015-25.

Castonguay Y, Laberge S, Nadeau P, Vezina L-P. 1994. A cold-induced gene from Medicago saliva encodes a bimodularprotein similar to developmentally regulated proteins. PlantMolecular Biology 24, 799-804.

CattiveUi L, Bartels D. 1990. Molecular cloning and character-ization of cold-regulated genes in barley. Plant Physiology93, 1504-10.

Chandler J, Dean C. 1994. Factors influencing the vernalizationresponse and flowering time of late flowering mutants ofArabidopsis thaliana (L) Heynh. Journal of ExperimentalBotany 45, 1247-88.

Chauvin L-P, Houde M, Sarhan F. 1993. A leaf-specific genestimulated by light during wheat acclimation to low temper-ature. Plant Molecular Biology 23, 255-65.

Chauvin L-P, Houde M, Sarhan F. 1994. Nucleotide sequenceof a new member of the freezing tolerance-associated proteinfamily-wheat. Plant Physiology 105, 1017-18.

Close TJ, Fenton RD, Yang A, Asghar R, DeMason DA, CroneDE, Meyer NC, Moonan F. 1993. Dehydrin: the protein. In:Close TJ, Bray EA, eds. Plant responses to cellular dehydrationduring environmental stress. USA: The American Society ofPlant Physiologists, 104-18.

Cobianchi F, Calvio C, Stoppini M, Buvoli M, Riva S. 1993.Phosphorylation of human hn RNP protein Al abrogates invitro strand annealing activity. Nucleic Acids Research4, 949-55.

Danyluk J, Houde M, Rassart E, Sarhan F. 1994. Differentialexpression of a gene encoding an acidic dehydrin in chilling-sensitive and freezing-tolerant gramineae species. FEBSLetters 344, 20-4.

Didierjean L, Frendo P, Burkard G. 1992. Stress responses inmaize: sequence analysis of cDNAs encoding glydne-richproteins. Plant Molecular Biology 18, 847-9.

Dolfcnis R, Jacobs M, Peacock WJ, Dennis ES. 1994.Differential interactions of promoter elements in stressresponses of the Arabidopsis Adh gene. Plant Physiology105, 1075-87.

Doll H, Haahr V, Segaard B. 1989. Relationships betweenvernalization requirement and winter hardiness in doubledhaploids of barley. Euphytica 42, 209 13.

Dreyfuss G, Matunis MJ, Pifiol-Roma S, Burd CG. 1993. hn


RNP proteins and the biogenesis of mRNA. Annual Reviewof Biochemistry 62, 289-321.

Dunn MA, Brown K, Lightowlers R, Hughes MA. 1996. A low-temperature-responsive gene from barley encodes a proteinwith single stranded nucleic acid binding activity which isphosphorylated in vitro. Plant Molecular Biology (in press).

Dunn MA, Goddard NJ, Zhang L, Pearce RS, Hughes MA.1994. Low-temperature-responsive barley genes have differentcontrol mechanisms. Plant Molecular Biology 24, 879-88.

Dunn MA, Hughes MA, Pearce RS, Jack PL. 1990. Molecularcharacterization of a barley gene induced by cold treatment.Journal of Experimental Botany 41, 1405-13.

Dunn MA, Hughes MA, Zhang L, Pearce RS, Quigley AS, JackPL. 1991. Nucleotide sequence and molecular analysis of thelow-temperature induced cereal gene, bit 4. Molecular andGeneral Genetics 229, 389-94.

Dunn MA, Morris A, Jack PL, Hughes MA. 1993. A low-temperature-responsive translation elongation factor 1 a frombarley (Hordeum vulgare L.). Plant Molecular Biology 23,221-5.

Gehring CA, Irving HR, Parish RW. 1990. Effects of auxin andabscisic acid on cytosolic calcium and pH in plant cells.Proceedings of National Academy of Sciences, USA 87,9645-9.

Gilmour SL, Tbomashow MF. 1991. Cold acclimation and cold-regulated gene expression in ABA mutants of Arabidopsisthaliana. Plant Molecular Biology 17, 1233-40.

Gilroy S, Jones RL. 1994. Perception of gibberellin and abscisicacid at the external face of the plasma membrance of barley{Hordeum vulgare L) aleurone protoplasts. Plant Physiology104, 1185-92.

Goddard NJ, Dunn MA, Zhang L, White AJ, Jack PL, HughesMA. 1993. Molecular analysis and spatial expression patternof a low-temperature-specific barley gene, bit 101. PlantMolecular Biology 23, 871-9.

Guo W, Ward RW, Thomashow MF. 1992. Characterization ofa cold-regulated wheat gene related to Arabidopsis cor 47.Plant Physiology 100, 915-22.

Hackett CA, Ellis RP, Forster BP, McNlcol JW, Macaulay M.1992. Statistical analysis of a linkage experiment in barleyinvolving quantitative trait loci for height and ear-emergencetime and two genetic markers on chromosome 4. Theoreticaland Applied Genetics 85, 120-6.

Hajela RK, Horvath DP, Gilmour SJ, Thomashow MF. 1990.Molecular cloning and expression of cor (cold-regulated)genes in Arabidopsis thaliana. Plant Physiology 93, 1246-52.

Hanvood JL, Jones AL, Perry HJ, Rutter AJ, Smith KL,Williams M. 1994. Changes in plant lipids during temperatureadaptation. In: Cossins AR, ed. Temperature adaptation ofbiological membranes. London, UK: Portland PressProceedings, 107-18.

Heino P, Sandman G, Lang V, Nordin K, Palva ET. 1990.Abscisic acid deficiency prevents development of freezingtolerance in Arabidopsis thaliana. Theoretical and AppliedGenetics 79, 801-6.

Heintzen C, Melzer S, Fischer R, Kappeler S, Apel K, StaigerD. 1994. A light- and temperature-entrained circadian clockcontrols expression of transcripts encoding nuclear proteinswith homology to RNA-binding proteins in meristematictissue. The Plant Journal 5, 799-813.

Hincha DK, Heber U, Schmitt JM. 1990. Proteins from frost-hardy leaves protect thylakoids against mechanical freeze-thaw damage in vitro. Planta 180, 416-19.

Hirose T, Sugita M, Sugiura M. 1993. cDNA structure,expression and nucleic acid-binding properties of three RNA-binding proteins in tobacco: occurrence of tissue-specificalternative splicing. Nucleic Acids Research 17, 3981-7.


Horvath D, McLarney BK, Thomashow MF. 1993. Regulationof Arabidopsis thaliana L. (Heyn) cor 78 in response to lowtemperature. Plant Physiology 103, 1047-53.

Houde M, Dhindsa RS, Sarhan F. 1992. A molecular marker toselect for freezing tolerance in Gramineae. Molecular andGeneral Genetics 234, 43-8.

Hughes MA, Dunn MA. 1990. The effect of temperature onplant growth and development. Biotechnology and GeneticEngineering Reviews 8, 161-88.

Hughes MA, Dunn MA, Pearce RS, White AJ, Zhang L. 1992.An abscisic acid-responsive, low temperature barley gene hashomology with a maize phospholipid transfer protein. Plant,Cell and Environment 15, 861-5.

Hughes MA, Pearce RS. 1988. Low temperature treatment ofbarley plants causes altered gene expression in shoot meris-tems. Journal of Experimental Botany 39, 1461-7.

Hugly S, Sommerville C. 1992. A role for membrane lipidpolyunsaturation in chloroplast biogenesis at low temper-ature. Plant Physiology 99, 197-202.

Jarillo JA, Capel J, Leyva A, Miguel J, Martinez-Zapater JM,Salinas J. 1994. Two related low-temperature-inducible genesof Arabidopsis encode proteins showing high homology to14-3-3 proteins, a family of putative kinase regulators. PlantMolecular Biology 25, 693-704.

Jarillo JA, Leyva A, Salinas J, Martinez-Zapater JM. 1993.Low temperature induces the accumulation of alcoholdehydrogenase mRNA in Arabidopsis thaliana, a chilling-tolerant plant. Plant Physiology 101, 833-7.

Knight MR, Campbell AK, Smith SM, Trewavas AJ. 1991.Transgenic plant aequorin reports the effects of touch, cold-shock and elicitors on cytoplasmic calcium. Nature 352,524-6.

Kodama H, Hamada T, Horiguchi G, Nishimura M, Iba K.1994. Genetic enhancement of cold tolerance by expressionof a gene for chJoroplast a>-3 fatty acid desaturase intransgenic tobacco. Plant Physiology 105, 601-5.

Kurkela S, Borg-Franck M. 1992. Structure and expression ofkin 2, one of two cold- and ABA-induced genes of Arabidopsisthaliana. Plant Molecular Biology 19, 689-92.

Kurkela S, Franck M. 1990. Cloning and characterization of acold- and ABA-inducible Arabidopsis gene. Plant MolecularBiology 15, 137-44.

Laberge S, Castonguay Y, Vezina L-P. 1993. New cold-anddrought-regulated gene from Medicago sativa. PlantPhysiology 101, 1411-12.

Lalk I, Dorffling K. 1985. Hardening, abscisic acid, proline andfreezing resistance in two winter wheat varieties. PhysiologiaPlantarum 63, 287-92.

Ling V, MSntyla E, Welin B, Sundberg B, Palva ET. 1994.Alterations in water status, endogenous abscisic acid contentand expression of rab 18 gene during the development offreezing tolerance in Arabidopsis thaliana. Plant Physiology104, 1341-9.

Lang V, Palva ET. 1992. The expression of a ra/vrelated gene,rab 18, is induced by abscisic acid during the cold acclimationprocess Arabidopsis thaliana (L.) Heyn. Plant MolecularBiology 20, 951 -62.

Law CN. 1966. The location of genetic factors affecting aquantitative character in wheat. Genetics 53, 487-98.

Leung J, Bouvier-Durand M, Morris P-C, Guerrier D, ChefdorF, Girandat J. 1994. Arabidopsis ABA response gene ABI 1:Features of a calcium-modulated protein phosphatase. Science264, 1448-52.

Lin C, Guo WW, Everson E, Thomashow MF. 1990. Coldacclimation in Arabidopsis and wheat. Plant Physiology94, 1078-83.


304 Hughes et al.

Lin C, Thomashow MF. 1992. A cold-regulated Arabidopsisgene encodes a polypeptide having potent cryo-protective activity. Biochemical and Biophysical ResearchCommunications 183, 1103-8.

Ludevid MD, Freire MA, Gomez J, Burd CG, Albetncio F,Giralt E, Dreyfuss G, Pages M. 1992. RNA bindingcharacteristics of a 16 kDa glycine-rich protein from maize.The Plant Journal 2, 999-1003.

Luo M, Lin L, Hill RD, Mohapatra SS. 1991. Primary structureof an environmental stress and abscisic acid-inducible alfalfaprotein. Plant Molecular Biology 17, 1267-9.

Lyons JM, Raison JK, Steponkus PL. 1979. The plant membranein response to low temperature: an overview. In: Lyons JM,Graham D, Raison JK, eds. Low temperature stress in cropplants. New York: Academic Press, 1-24.

MantylS E, Lang V, Palva ET. 1995. Role of abscisic acid indrought-induced freezing tolerance cold acclimation andaccumulation of LT1 78 and RAB 18 proteins in Arabidopsisthaliana. Plant Physiology 107, 141-8.

Meyer K, Leube MP, Grill E. 1994. A protein phosphatase 2Cinvolved in ABA signal transduction in Arabidopsis thaliana.Science 264, 1452-5.

Miguel M, Jas Jr D, Dooner H, Browse J. 1993. Arabidopsisrequires polyunsaturated lipids for low-temperature survival.Proceedings of the National Academy of Sciences, USA90, 6208-12.

Molina A, Garcia-Olmedo F. 1993. Developmental and patho-gen-induced expression of three barley genes encoding lipidtransfer proteins. The Plant Journal 4, 983-91.

Monroy AF, Castonguay Y, Laberge S, Sarhan F, Vezina LP,Dhindsa RS. 1993a. A new cold-induced alfalfa gene isassociated with enhanced hardening at subzero temperature.Plant Physiology 102, 873-9.

Monroy AF, Sartian F, Dhindsa RS. 19936. Cold-inducedchanges in freezing tolerance, protein phosphorylation andgene expression. Plant Physiology 102, 1227-35.

Mundy J, Rogers JC. 1986. Selective expression of a probableamylase/protease inhibitor in barley aleurone cells: compar-isons to the barley amylase/subtillism inhibitor. Planta169, 51-63.

Murata N, Ishizaki-Nishizawa O, Higashi S, Hayashi H, TasakaY, Nishida I. 1992. Genetically engineered alteration in thechilling sensitivity of plants. Nature 356, 710-13.

Neven LG, Haskell DW, Hofig A, Li Q-B, Guy CL. 1993.Characterization of a spinach gene responsive to lowtemperature and water stress. Plant Molecular Biology21, 291-305.

Nordin K, Heino P, Palva ET. 1991. Separate signal pathwaysregulate the expression of a low-temperature-induced gene inArabidopsis thaliana. Plant Molecular Biology 16, 1061-71.

Nordin K, Vahala T, Palva ET. 1993. Differential expression oftwo related, low-temperature-induced genes in Arabidopsisthaliana. Plant Molecular Biology 21, 641-53.

Orr W, Iu B, White TC, Robert LS, Singh J. 1992.Complementary DNA sequence of a low-temperature-inducedBrassica napus gene with homology to the Arabidopsis thalianakin 1 gene. Plant Physiology 98, 1532^4.

Ouellet F, Houde M, Sarhan F. 1993. Purification, characteriza-tion and cDNA cloning of the 200 kDa protein induced bycold acclimation in wheat. Plant Cell Physiology 34, 59-65.

Palta JP, Whitaker BD, Weiss LS. 1993. Plasma membranelipids associated with genetic variability in freezing toleranceand cold acclimation of Solanum species. Plant Physiology103, 793-803.

Pan A, Hayes PM, Chen F, Chen THH, Blake T, Wright S,Karsai I, Beds Z. 1994. Genetic analysis of the components

of winter hardiness in barley (Hordeum vulgare L.). Theoreticaland Applied Genetics 89, 900-10.

Pardossi A, Vemieri P, Mori B, Toguoni F. 1994. Water relationsand ABA in chilled plants. In: DSrffling K, Brettschneider B,Tantau H, Pithan K, eds. Crop adaptation to cool climates.ECSP-EEC-EAEC, Brussels, 215-22.

Pearce RS, Dunn MA, Rixon JA, Harrison P, Hughes MA.1996. Expression of cold-inducible genes and frost hardinessin the crown meristem of young barley (Hordeum vulgare L.cv. Igri) plants grown in different environments. Plant, Celland Environment (in press).

Plaschke J, B6rner A, Xie DX, Koebner RMD, Schlegel R, GaleMD. 1993. RFLP mapping of genes affecting plant heightand growth habit in rye. Theoretical and Applied Genetics85, 1049-54.

Pyee J, Yu H, Kolattukudy PE. 1994. Identification of a lipidtransfer protein as the major protein in the surface wax ofbroccoli (Brassica oleracea) leaves. Archives of Biochemistryand Biophysics 311, 460-8.

Pyee J, Kolattukudy PE. 1995. The gene for the major cuticularwax-associated protein and three homologous genes frombroccoli (Brassica oleracea) and their expression patterns. ThePlant Journal 7, 49-59.

Roberts DWA. 1990. Identification of loci on chromosome 5Aof wheat involved in control of cold hardiness, vernalization,leaf length, rosette growth habit, and height of hardenedplants. Genome 33, 247-59.

Rock CD, Quatrano RS. 1994. Insensitivity is in the genes.Current Biology 4, 1013-15.

Roughan PG. 1985. Phosphatidylglycerol and chilling sensitivityin plants. Plant Physiology 11, 740-6.

Sa'ez-Va'squez J, Raynal M, Meza-Basso T, Delseny M. 1993.Two related, low-temperature-induced genes from Brassicanapus are homologous to the human tumour bbc 1 (breastbasic conserved) gene. Plant Molecular Biology 23, 1211-21.

Smith H. 1990. Signal perception, differential expression withinmultigene families and the molecular basis of phenotypicplasticity. Plant, Cell and Environment 13, 585-94.

Steponkus PL, Uemura M, Bateamo RA, Arvinte T, Lynch DV.1988. Transformation of the cryobehaviour of rye protoplastsby modification of the plasma membrane lipid composition.Proceedings of the National Academy of Sciences, USA85, 9026-30.

Steponkus PL. 1984. Role of the plasma membrane in freezinginjury and cold acclimation. Annual Review of PlantPhysiology 35, 543-84.

Sturm A. 1992. A wound-inducible glycine rich protein fromDaucus carota with homology to single-stranded nucleic acid-binding proteins. Plant Physiology 99, 1689-92.

Sutka J, Galiba G, Quarrie SA, Snape JW. 1996. Cytogeneticstudies on frost resistance in wheat (Triticum aestivum L.).In: Crop adaptation to cool climates. EU: COST 814 (in press).

Sutka J, Snape JW. 1989. Location of a gene for frost resistanceon chromosome 5A of wheat. Euphvtica 42, 41-4.

Sutton F, Ding X, Kenefick DG. 1992. Group 3 LEA gene HVA1 regulation by cold acclimation and deacclimation in twobarley cultivars with varying freeze resistance. PlantPhysiology 99, 338-40.

Takahashi R, Yasuda S. 1970. Genetics of earliness and growthhabit in barley. In: Milan RA, ed. Barley genetics, Vol. II.Proceedings of the 2nd International Barley GeneticsSymposium, Washington State University Press, 388-408.

Welln BV, Olson A, Nylander M, Palva ET. 1994.Characterization and differential expression of dhnjleajrab-like genes during cold acclimation and drought stress inArabidopsis thaliana. Plant Molecular Biology 26, 131-44.


Weretilnyk E, Orr W, White TC, Lu B, Singh J. 1993.Characterization of three related low-temperature-regulatedcDNA's from winter Brassica napus. Plant Physiology 101,171-7.

White AJ, Dunn MA, Brown K, Hughes MA. 1994. Comparativeanalysis of genomic sequence and expression of a lipidtransfer protein gene family in winter barley. Journal ofExperimental Botany 45, 1885-92.

White TC, Simmonds D, Donaldson P, Singh J. 1994. Regulationof BN 115, a low-temperature-responsive gene from winterBrassica napus. Plant Physiology 106, 917-28.

Wilhelm KS, Thomashow MF. 1993. Arabidopsis thaliana cor15b, an apparent homologue of cor 15a, is strongly responsiveto cold and ABA, but not drought. Plant Molecular Biology23, 1073-7.

Williams ME, Foster R, Chua N-H. 1992. Sequences flankingthe hexameric G-box core CACGTG affect the specificity ofprotein binding. The Plant Cell 4, 485-96.

Wolfraim LA, Langis R, Tyson H, Dhindsa RA. 1993. cDNAsequence, expression and transcript stability of a coldacclimation-specific gene, cas 18, of alfalfa (Medicago falcata)cells. Plant Physiology 101, 1275-82.

Wolter FP. Schmidt R, Heinz E. 1992. Chilling sensitivity of


Arabidopsis thaliana with genetically engineered membranelipids. EM BO Journal 11, 4685-92.

Wu J, Browse J. 1995. Elevated levels of high-melting-pointphosphatidylglycerols do not induce chilling sensitivity in anArabidopsis mutant. The Plant Cell 7, 17-27.

Xin ZY, Law CN, Worland AJ. 1988. Studies on the effects ofthe vernalization-responsive genes on the chromosomes ofhomeologous group 5 of wheat. In: Proceedings of the 7thinternational wheat genetics symposium, Vol. 1. Cambridge,UK: Institute of Plant Science Research, 675-80.

Yamaguehi-Shinozaki K, Shinozaki K. 1994. A novel cw-actingelement in an Arabidopsis gene is involved in responsivenessto drought, low-temperature, or high-salt stress. The PlantCell 6, 251-64.

Zhang L, Dunn MA, Pearce RS, Hughes MA. 1993. Analysisof organ specificity of a low-temperature-responsive genefamily in rye (Secale cereale L). Journal of ExperimentalBotany 44, 1787-93.

Zhu B, Chen THH, Li PH. 1993. Expression of an ABA-responsive osmotin-like gene during the induction of freezingtolerance in Solanum commersonii. Plant Molecular Biology21, 729-35.


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The molecular biology of plant acclimation to low temperature