The CBF1-dependent low temperature signalling … BHALERAO 3, NORMAN P. A. HUNER 4, CHAD E. FINN 2,5, TONY H. H. CHEN 2 & VAUGHAN HURRY 1 1Umeå Plant Science Centre, Department of

Plant Cell and Environment

(2006)

29

1259ndash1272 doi 101111j1365-3040200601505x

copy 2006 The AuthorsJournal compilation copy 2006 Blackwell Publishing Ltd

1259

The CBF regulon in

Populus

sppC Benedict

et al

Correspondence Vaughan Hurry Fax

+

46 90 7866676 e-mailVaughanHurryplantphysumuse

The CBF1-dependent low temperature signalling pathway regulon and increase in freeze tolerance are conserved in

Populus

spp

CATHERINE BENEDICT

1

JEFFREY S SKINNER

2

RENGONG MENG

2

YONGJIAN CHANG

2

RISHIKESH BHALERAO

3

NORMAN P A HUNER

4

CHAD E FINN

25

TONY H H CHEN

2

amp VAUGHAN HURRY

1

1

Umearing Plant Science Centre Department of Plant Physiology Umearing University S-901 87 Umearing Sweden

2

Department of Horticulture Oregon State University Corvallis Oregon 97331-7304 USA

3

Umearing Plant Science Centre Department of Forest Genetics Swedish University of Agricultural Sciences S-901 83 Umearing Sweden

4

Department of Biology and Biotron University of Western Ontario London N6A 5B7 Ontario Canada and

5

US Department of Agriculture-Agricultural Research Service Corvallis Oregon 97330 USA

ABSTRACT

The meristematic tissues of temperate woody perennialsmust acclimate to freezing temperatures to survive the win-ter and resume growth the following year To determinewhether the C-repeat binding factor (CBF) family of tran-scription factors contributing to this process in annual her-baceous species also functions in woody perennials weinvestigated the changes in phenotype and transcript profileof transgenic

Populus

constitutively expressing

CBF1

from

Arabidopsis

(

AtCBF1

) Ectopic expression of

AtCBF1

was sufficient to significantly increase the freezing toleranceof non-acclimated leaves and stems relative to wild-typeplants cDNA microarray experiments identified genes up-regulated by ectopic

AtCBF1

expression in

Populus

dem-onstrated a strong conservation of the CBF regulonbetween

Populus

and

Arabidopsis

and identified differ-ences between leaf and stem regulons We studied theinduction kinetics and tissue specificity of four

CBF

paral-ogues identified from the

Populus balsamifera

subsp

tri-chocarpa

genome sequence (

PtCBF

s) All four

PtCBF

s arecold-inducible in leaves but only

PtCBF1

and

PtCBF3

show significant induction in stems Our results suggest thatthe central role played by the CBF family of transcrip-tional activators in cold acclimation of

Arabidopsis

hasbeen maintained in

Populus

However the differentialexpression of the

PtCBF

s and differing clusters of CBF-responsive genes in annual (leaf) and perennial (stem)tissues suggest that the perennial-driven evolution of winterdormancy may have given rise to specific roles for theselsquomaster-switchesrsquo in the different annual and perennialtissues of woody species

Key-words

cold tolerance microarray

INTRODUCTION

Adaptation to low temperatures is one of the most impor-tant components in the evolutionary process in temperateand boreal tree species (Saxe

et al

2001) Even when non-lethal the accumulated lifetime costs of recurring freezedamage to tree fitness are significant spring frosts damageflushing buds and flowers decreasing tree growth and seedproduction (Selas

et al

2002) and leaves prematurely lostto autumn frosts reduce the ability of the tree to coldharden (Howell amp Stockhause 1973) Under field condi-tions woody perennials develop deep winter hardiness inresponse to two environmental cues (Weiser 1970) Short-ening day length first initiates the transition from activegrowth to winter dormancy which may take many weeksto complete (Weiser 1970 Fuchigami Weiser amp Evert 1971Junttila 1976) The onset of winter dormancy results in amoderate increase in freezing tolerance (FT) but subse-quent exposure to low temperatures is required to promotethe deep winter hardiness that is unique to woody perenni-als (Weiser 1970 Fuchigami

et al

1971) Woody perennialslike herbaceous species can also acquire cold tolerancewhen exposed to low temperatures during long days butfull winter hardiness only develops under the combinedstimuli of short photoperiods and low temperatures (Chris-tersson 1978 Li

et al

2002 2003a Puhakainen

et al

2004)The molecular mechanisms governing the acquisition of

FT are largely unknown in woody plants The accumulationof dehydrins has been associated with the development ofFT in a number of woody species (Arora amp Wisniewski1994 Arora Wisniewski amp Rowland 1996 CampalansPages amp Messeguer 2000 Richard

et al

2000) and have beenshown to correlate with seasonal variations in FT (Arora

et al

1996 Wisniewski

et al

1996 Welling Kaikuranta ampRinne 1997 Rinne Welling amp Kaikuranta 1998 Lim Krebsamp Arora 1999) However although significant efforts havebeen made to assign roles to different dehydrins (Wis-niewski

et al

1999 Bravo

et al

2003) their biochemical

1260

C Benedict

et al



29

1259ndash1272

functions remain largely unknown (Svensson

et al

2002)Cold acclimation is better understood in herbaceous annu-als such as

Arabidopsis thaliana

where members of theC-repeat binding factor (CBF or DREB1) family of tran-scriptional activators which bind the

cis

-element known asthe C-repeat (CRT)dehydration-responsive element(DRE) (Stockinger Gilmour amp Thomashow 1997) havebeen shown to control the transcription of a suite of genesthat play important roles in cold acclimation and the devel-opment of FT The

Arabidopsis

CBFDREB1 family con-sists of six paralogues but only three [

CBF1

(

DREB1b

)

CBF2

(

DREB1c

) and

CBF3

(

DREB1a

)] are cold-induc-ible (Sakuma

et al

2002) These three low-temperature-responsive

CBF

s colocalize to an 87 kb region of chromo-some 4 and share a conserved AP2 DNA binding regionflanked by the characteristic amino acid motifsPKKPAGRxKFxETRHP and DSAWR (Jaglo

et al

2001)Based on a number of experiments using transgenic plantsat least 12 of the cold-induced transcriptional changesobserved in

Arabidopsis

are accounted for by the membersof the

CBF

transcription factor family (

CBF1-3

) (Fowleramp Thomashow 2002 Vogel

et al

2005 van Buskirk amp Tho-mashow 2006) and ectopic expression of

Arabidopsis

CBF1-4 has been shown to improve FT of non-acclimatedplants (Jaglo-Ottosen

et al

1998 Gilmour

et al

2000Haake

et al

2002 Gilmour Fowler amp Thomashow 2004)The accumulation of CBF transcripts and the activity of

the CRT regulatory motif in

Arabidopsis

is also modulatedby the presence and quality of light during cold stress (Kim

et al

2002 Fowler Cook amp Thomashow 2005) A shortperiod of exposure to red light has been shown to be suffi-cient to induce CRT-driven GUS reporter expression in thecold and this induction is photo-reversible with far-redlight GUS reporter expression is also altered in phyto-chrome mutant backgrounds suggesting that day lengthmediates expression of the CBF regulon through phyto-chrome B in

Arabidopsis

(Kim

et al

2002) Furthermorethe kinetics of endogenous CBF transcript accumulation isreduced in complete darkness (Kim

et al

2002) suggestingthat light regulation occurs at a point upstream of CBFexpression These data raise the question of whether CBFor related transcriptional regulators may be involved in theday length-mediated growth cessation in woody species Ithas been suggested that growth cessation in woody peren-nials mediated by short days and phytochrome operatesindependently of the low-temperature-driven acquisition ofdeep FT (Welling

et al

2002) However the observationthat pre-treatment with short days enhances the expressionof low temperature response genes (Puhakainen

et al

2004) and the finding that a CBF-like transcription factoris induced by short days in the cambial meristem of poplar(Schrader

et al

2004) indicates that there may be cross-talkbetween these two acclimation mechanisms

Following their original discovery in

Arabidopsis

the listof plants with cold-inducible

CBF

orthologues hasexpanded to include cold-sensitive and cold-tolerantannual species (Jaglo

et al

2001) and woody perennials(Kitashiba

et al

2002 Owens

et al

2002) Puhakainen

et al

(2004) have also demonstrated that AtCBF3 can activategene transcription via the birch

Bplti36

promoter sequencein transgenic

Arabidopsis

indicating that the structure andfunction of the active domain in the promoters of CBF-mediated cold-responsive genes have been conserved inherbaceous and woody perennials However detailed stud-ies of the role of CBF-related proteins in cold acclimationin tree species have been lacking Based on the demon-strated effects of both low temperature and light on CBFtranscript accumulation in

Arabidopsis

we have examinedhow CBF expression affected the FT of non-dormant pop-lar leaf and meristematic stem tissue under long-day growthconditions This experimental design enabled us to focus onthe role the CBF-mediated signalling pathway plays in thetemperature response of different tissues in a woody peren-nial without complicating interactions with day length anddormancy Our data demonstrate (1) that CBFs areinvolved in the FT mechanism of temperate trees speciessuch as poplar (2) that distinct regulons control stem (mer-istematic) versus leaf (non-meristematic) FT and (3) thatthe

Populus

genome contains multiple CBFs that are likelyto be differentially employed in perennial (stem) andannual (leaf) tissues

MATERIALS AND METHODS

Construction of transformation vector

A CaMV 35S

Promoter

Arabidopsis

CBF1 cDNA35S

Terminator

cassette (Jaglo-Ottosen

et al

1998) was ligated as a

Hind

IIIfragment into the binary vector pGAH (Onouchi

et al

1991) that had been linearised with

Hind

III

Poplar transformation

Plants of poplar clone 717-1B4 (

Populus tremula

times

alba

)were grown

in vitro

in a culture room at 24

deg

C under cool-white fluorescent lights (40ndash60

micro

mol m

minus

2

s

minus

1

16 h photope-riod) Stem internodes (7ndash10 mm in length) and leaf discs(5 mm in diameter) from fully expanded young leaves wereused as the explants for the transformation (DeBlock1990)

Agrobacterium tumefaciens

strain EHA105 carrying theplasmid pGAH35SAtCBF1 was grown overnight at 28

deg

Cin liquid YEP medium supplemented with 50 mg L

minus

1

hygro-mycin and 50 mg L

minus

1

kanamycin The cells were collectedby centrifugation at 1500 g for 20 min and resuspended toan OD

600

=

04ndash06 in Murashige and Skoog (MS) mediumwith 20

micro

g L

minus

1

acetosyringone (AS) Explants (stem intern-odes or leaf discs) were soaked for 10ndash20 min in the bacte-rial suspension incubated on a shaker (300 g) for 1 h atroom temperature followed by cultivation on callus induc-tion medium (CIM pH 52) supplemented with 20

micro

g L

minus

1

AS at 25

deg

C in the dark for 2ndash3 d After 2ndash3 d of cocultiva-tion explants were washed four times with double-distilledwater and once with wash solution (WS pH 58) supple-mented with 500 mg L

minus

1

cefotaxime The washed explantswere blotted dry with paper towels allowed to stand in a

The CBF regulon in

Populus

spp

1261



29

1259ndash1272

laminar flow hood for drying for about 30 s and then trans-ferred to CIM supplemented with 250 mg L

minus

1

cefotaximeand 50 mg L

minus

1

kanamycin for 2 weeks at 25

deg

C in darknessExplants were transferred to shoot induction medium(SIM pH 58) supplemented with 50 mg Lminus1 kanamycinand 250 mg Lminus1 cefotaxime to induce shoot formation for2ndash3 weeks Explants were then transferred to shoot devel-opment and elongation medium (SEM pH 58) supple-mented with 50 mg Lminus1 kanamycin and 250 mg Lminus1

cefotaxime until shoots were well developed (gt 1 cm inlength) During this period the explants were transferredto fresh SEM supplemented with 50 mg Lminus1 kanamycin and250 mg Lminus1 cefotaxime every two weeks Regeneratedshoots (gt 10 cm) were excised from the explants and fur-ther screened for kanamycin resistance on rooting medium(RM pH 58) supplemented with 50 mg Lminus1 kanamycin and250 mg Lminus1 cefotaxime Leaves from the rooted plants werecollected for DNA extraction for PCR to verify the pres-ence of CaMV 35S promoterAtCBF1 transgene in thepoplar genome and for RNA extraction for Northern blotanalysis to verify lines expressing detectable levels of thetransgene

Leaf and stem FT assays

Plants of wild-type (WT) and transgenic lines 1 and 2 werepropagated by cuttings in vitro and rooted plants weretransplanted into 1 gallon pots containing Sunshine SB40Professional Growing Mix (Sun Gro Horticulture IncBellevue WA) supplemented with 7 pumice (weightvol-ume) and 014 perlite and fertilized with 004 osmo-cote (The Scotts Company Marysville OH) Plants weregrown in a greenhouse maintained at a 16 h day (25 plusmn 3 degC)and 8 h night (20 plusmn 3 degC) with supplemental lighting (mer-cury halide lamps providing 400ndash500 micromolm2 sminus1) After6 weeks of growth plants of both transgenic lines and WTwere divided into two groups One group of plants wastransferred to a cold room (2 degC 16 h photoperiod) for1 week while the other group was maintained in the green-house under the greenhouse conditions mentioned earlierFor freezing tests tissue samples 1-cm-diameter leaf discswere excised from each side of the two to three uppermostfully expanded leaves and 2 cm stem segments were har-vested Three leaf discs or stem segments were used perassay and FT was determined by ion leakage (Sukumaranamp Weiser 1972)

RNA extraction for microarray analysis

Leaf and stem samples were collected from three WTplants and three individuals from each transgenic line (1and 2) grown under either the warm greenhouse conditionsdiscussed earlier or after 1 week of cold acclimation (2 degCwith 16 h photoperiod) Total RNA was extracted fromfrozen stem and leaf samples (Hughes amp Galau 1988) theRNA samples were reconstituted in TE buffer and the totalRNA was further purified using QIAGEN RNeasy columns(Qiagen Valencia CA USA) Aliquots of these total RNA

preparations from each clonally propagated genetic back-ground were then pooled before being used for fluorescentprobe synthesis This experimental protocol was repeatedon clonally propagated material grown at a later dateresulting in two independent biologically replicatedexperiments

Preparation of fluorescent targets

Targets were labelled indirectly by incorporating aminoal-lyl-dUTPs (Sigma St Louis MO USA Pharmacia Fair-field CT USA) and subsequently coupling with either Cy3or Cy5 dye The reverse transcription reaction mixture(30 microL) contained 20 microg total RNA with 5 microg of oligo(dT)20 mer 10 mM DTT 500 microM each of dATP dCTP anddGTP 300 microM dTTP 200 microM aa-dUTP 30ndash40 units RNaseinhibitor and 200 units of SuperScript II reverse tran-scriptase (Life Technologies Grand Island NY) in 1XSuperscript first-strand buffer (50 mM Tris-HCl pH 8375 mM KCl 3 mM MgCl2 and 20 mM DTT) (Life Technol-ogies) After incubation at 42 degC for 2 h the reaction prod-ucts were treated with 10 microL 1 M NaOH 10 microL 05 MEDTA incubated at 65 degC for 15 min and neutralized with50 microL 1 M HEPES (pH 70) Samples were purified onQIAGEN MinElute Clean-up columns (Qiagen) asdirected except that the final elution was performed with15 microL of NaHCO3 pH 90 into an amber eppendorf con-taining the appropriate vacuum-dried Cy dye pellet [17 ofthe commercial aliquot (Amersham Pharmacia Biotech1 mg protein-labelling aliquots)] The eppendorf contentswere mixed until the Cy dye pellet was fully resuspendedspun down and incubated in darkness at room temperaturefor 2 h At the end of the incubation period the labellingmixture was diluted to 100 microL using sterile water andQIAGEN PCR purification columns (Qiagen) were used asdirected except that the final elution was performed using30 microL sterile water Respective Cy3 and Cy5 samples forcomparison were combined and reduced under vacuum toa volume of 13 microL at 40 degC This probe mixture was com-bined with 05 microL of 25 microg microLminus1 yeast tRNA 20 microL of10 microg microLminus1 oligo(dA) 60 microL of 20X SSC 75 microL of deonizedformamide and 10 microL 10 sodium dodecyl sulphate (SDS)and denatured at 95 degC for 1 min After 2 min cooling onice the prepared probe mixture was used for hybridization

Microarray hybridization and scanning

Populus 13K duplicate array slides (Andersson et al 2004)were prehybridized and hybridized using the solutions andprotocols reported previously (Hertzberg et al 2001) Theslides were scanned with a ScanArray Lite scanner (PerkinElmer Wellesley MA USA) and ScanArray express soft-ware Separate images were acquired for each fluor at aresolution of 10 microm per pixel Scanning photomultiplierand laser power settings were optimized by the software tominimize saturated spots and maximize the signal-to-noiseratio Microarray spots were flagged and quantified usingGenepix 50 (Axon Instruments Foster City CA USA)

1262 C Benedict et al

copy 2006 The AuthorsJournal compilation copy 2006 Blackwell Publishing Ltd Plant Cell and Environment 29 1259ndash1272

software The resulting mean signal and medianbackground data for each channel and spot were importedand normalized using the Bioconductor package(wwwbioconductororg) applying Edwards backgroundcorrection and loess normalizations within individualarrays and Aquartile between-array normalization beforeimport into Genespring 60 (Silicon Genetics RedwoodCity CA USA) for visualization Gene lists for each com-parison (Supplemental Tables) were constructed from thedata collected from four hybridizations of each line(including two independent biological replicates and dye-swapping) Duplicate printing of probes on the hybridizedUPSCndashKTH 13K Populus slides also increased the totalreplication twofold for each probe Therefore the finalgene lists (incorporating expression data from both trans-genic lines) represent the mean expression change and sta-tistical data from 16 replicates We set statisticalsignificance at P lt 005 plus an arbitrary fold-change (FC)cut-off of FC gt 175X to construct the gene lists The rawand normalized microarray data are publicly available athttpwwwupscbasedbumuse

Multiplex and real-time RT-PCR transcript quantification

Total RNA was isolated from 50 to 100 mg of powder fromleaf or stem tissue from 6-week-old hybrid aspen (Populustremula times tremuloides clone T89) trees using Trizol reagent(Gibco BRL Paisley UK) according to the manufacturerrsquosinstructions except for an extra 10 min centrifugation stepat 4 degC after the addition of Trizol the use of 05 volumesof bromochloropropane in place of 1 volume of chloroformduring phase separation and use of 11 isopropanolhighsalt solution (08 M sodium citrate 12 M sodium chloride)in place of isopropanol during RNA precipitationQIAGEN RNeasy columns (Qiagen) were used as directedto purify samples Three micrograms total RNA was usedin reverse transcription with the First Strand cDNA kit(Amersham) according to the manufacturers instructionsSemi-quantitative multiplex PCR was used to determinerelative amounts of AtCBF1 transcript in the transgenicPopulus lines using the Cbf-f (ACGAATCCCGGAGTCAACATGC) and Cbf-r (ccttcgctctgttccggtgtataaat) gene-specific primers and Universal 18S (Quantum RNA 18Sstandards Ambion Austin TX USA) primers with com-petimer (28 ratio) as the endogenous standard Real-timePCR quantification was performed using iQ SYBR greensupermix (Bio-Rad Hercules CA USA) as instructed withgene-specific primers [PtCBF1F ACACAGGATGCCTTGTTTCC PtCBF1R GGAGTTCAACCAGGTGCAAT PtCBF2F GGGAGGTGAGTTGATGAGGAPtCBF2R TATTAGCCAACAACCCTGGC PtCBF3FTTTCAAATGAGGCCAAGGAC PtCBF3R CCTCCCTCCTGAAATCTTCC PtCBF4F GGCAGCAAATGAGGCAGCAG PtCBF4R CTTGAGCAATCCTCTAGCACTGCAT Universal 18S rRNA primers (Ambion)and sim2 ng cDNA template Relative PtCBF abundance wasquantified and then normalized using the ∆∆ct method of

reference gene 18S calibrator sample 2 ng 3 h cold-treated leaf cDNA]

RESULTS

Ectopic expression of AtCBF1 in Populus

Arabidopsis CBF paralogues have been used to manipulateFT in a number of herbaceous plant species (Jaglo et al2001 Hsieh et al 2002 Lee et al 2003 2004) To testwhether a CBF regulon exists and plays a role in the FT ofoverwintering woody perennials we generated transgenicPopulus tremula times alba (clone 717-1B4) ectopicallyexpressing the well-characterized AtCBF1 gene fromArabidopsis (Fig 1a) We generated 19 independent trans-genic lines expressing AtCBF1 and characterized two ofthese lines expressing AtCBF1 at rates equivalent to endog-enous AtCBF1 transcript levels in Arabidopsis after 3 hcold treatment as assessed by RT-PCR (Fig 1b amp c) Theselected lines with high AtCBF1 transcript abundance(AtCBF1-Poplar) had fewer roots and slower growth rateswhen cultivated in vitro(Fig 2a) but fully recovered after3ndash6 weeks growth in soil (Fig 2bndashd)

AtCBF1-Poplar lines have improved leaf and stem FT

The effect of ectopic expression of AtCBF1 in poplar onFT of leaves and stems was tested using an electrolyteleakage test This assay provides an estimate of cell dam-age (Dexter Tottingham amp Garber 1932 Sukumaran ampWeiser 1972) and is quantified by determining the temper-ature leading to leakage of 50 of cellular electrolytes(TEL50) Non-acclimated leaves from both AtCBF1-Poplarlines showed a significant (P lt 0001) gain in FT fromminus39 degC for non-acclimated WT to an average of minus69 degC forAtCBF1-Poplar (Table 1) Ectopic expression of AtCBF1also significantly improved stem FT in non-acclimatedplants (TEL50 minus54 degC for the AtCBF1 lines versus minus41 degCfor WT P lt 0001) (Table 1) These FT data demonstrate

Table 1 TEL50 measurements for leaf and stem tissues of non-acclimated (NA) and 1 week cold-acclimated (CA) hybrid aspen trees Values represent the mean of six independent experiments plusmn standard error Statistical significance was tested first by one-way ANOVA with Dunnettrsquos post-test using GraphPad Prism version 400 for Windows (GraphPad Software San Diego CA USA)

Leaves Stems

NA CA NA CA

TEL50

(degC)WT minus39 plusmn 02 minus173 plusmn 12 minus41 plusmn 03 minus99 plusmn 02Line 1 minus67 plusmn 10 minus183 plusmn 11 minus52 plusmn 04 minus107 plusmn 10Line 2 minus71 plusmn 07 minus187 plusmn 25 minus57 plusmn 06 minus113 plusmn 17

WT wild typeP lt 0001

The CBF regulon in Populus spp 1263


that the cryoprotective benefit of ectopic AtCBF1 expres-sion shown in Arabidopsis leaves (Jaglo-Ottosen et al1998 Gilmour et al 2000) extends to leaves and stemsof poplar and suggests that Populus spp utilize a CBF-mediated signalling pathway to control at least part ofthe cold acclimation response

The AtCBF1 regulon(s) in Populus

To determine whether AtCBF1-driven transcriptionalchanges mirrored the endogenous transcriptional responseto low temperature the transcriptomes of WT warm-grownleaf and stem tissue were compared with the transcriptomesof leaf and stem tissues of warm-grown poplar ectopicallyexpressing AtCBF1 and with the transcriptomes of leaf andstem tissues from WT trees after 7 d of cold acclimation at2 degC The cDNA microarray experiments utilized mRNAfrom WT plants and the two independent AtCBF1-Poplarlines and gene lists were constructed using the averageresult of two independent growth experiments Theseexperiments enabled us to determine (1) which genes weresusceptible to AtCBF1 regulation (2) whether the genesthat respond to AtCBF1 control showed differentialexpression in the meristematic stem tissues versus leaf tis-sue and (3) how much overlap there was between the CBFregulon and the endogenous (untransformed) response ofpoplar leaves and stems to cold

Using our cut-offs (FC gt18 P lt 005) we identified 63genes up-regulated in leaves from warm (23 degC) grownAtCBF1-Poplar relative to leaves from warm-grown WTplants (Table S1a) Almost half of the total list (2963) ofAtCBF1-Poplar leaf regulon members was composed ofnovel lsquoexpressed proteinsrsquo and other proteins with noknown function Of the 34 AtCBF1-Poplar leaf regulongenes annotated to have a known function more thanone-third (35) encoded metabolic enzymes Togetherthe classes lsquoCell rescuersquo (15) and lsquoTranscriptionrsquo (21)made up another third with the remaining classes repre-sented being lsquoDevelopmentrsquo (9) lsquoCellular communica-tionsignal transductionrsquo (6) lsquoStoragersquo (6) lsquoCellularbiogenesisrsquo (6) and lsquoProtein fatersquo (3) (Fig 3) Thisfunctional distribution of genes in the AtCBF1-Poplar leafregulon mirrored that of the genes endogenously up-regu-lated in WT leaves by 7 d at low temperature (Fig 3Table S2) In contrast the functional class lsquoTranscriptionrsquomade up the dominant fraction (41) of the AtCBF1-Poplar regulon in warm-grown perennial stem tissue fol-lowed by lsquoCellular communicationsignal transductionrsquo(18) lsquoCell rescuersquo (12) and lsquoMetabolismrsquo (12)(Fig 3)

The functional analysis of the genes induced by 7 d ofcold (2 degC) treatment in WT Populus leaves and stemsshowed that these tissue-specific regulons were not asinnately different as the AtCBF1-Poplar leaf and stem reg-

Figure 1 Expression of the AtCBF1 transgene (a) pGAH35SAtCBF1 expression construct (b) Relative expression of the AtCBF1 gene in the stem (S) and leaf (L) tissue of wild-type Populus (PtWT) AtCBF1-Poplar line 1 AtCBF1-Poplar line 2 grown for 6 weeks in the greenhouse at 23 degC and in warm (23 degC) grown wild-type Arabidopsis Col 0 (AtWT) after 3 h exposure to 5 degC respectively (c) Semi-quantitative multiplex RT-PCR analysis was performed on first-strand cDNA generated from total RNA extracted from wild-type and transgenic plants 6 weeks after transfer to soil in the greenhouse Differences in template starting quantity in the wild-type and transgenic lines were corrected by normalizing to the expression of the 18S rRNA (18S) in the respective samples Error bars indicate standard errors of six independent experiments

pGAH35SAtCBF1

35SP AtCBF1NPTII (Km )R NOST

LBRB

35SPNOSp HygR

NOST NOST

AtCBF118S

S L S L S L

Rel

ativ

em

RN

Aco

nten

tC

BF

118

S

S L

PtWT Line 1 Line 2

S LS L0

1

2

3

4L

L

AtWT

(a)

(b)

(c)



ulon data (Fig 3) This comparison held true even at thelevel of individual genes more than half (57) of the up-regulated genes with known function from WT cold-treatedstem tissue were also significantly up-regulated in WT cold-treated leaf tissue In fact in terms of functional composi-tion the AtCBF1-Poplar leaf WT cold leaf and WT coldstem regulons showed high similarity while the AtCBF1-Poplar stem regulon differed largely on the basis of a sub-stantial depletion of metabolic genes (12 versus 35 inAtCBF1-Poplar leaf tissue and 38 in WT cold stem) andan enrichment of genes regulating transcription (41 ver-sus 21 in AtCBF1-Poplar leaf tissue and 22 for WT coldstem) (Fig 3)

As indicated by the functional analysis the normalizedexpression data for all genes significantly up-regulated byAtCBF1 in at least one tissue showed that the leaf(Table S1a) and stem (Table S1b) AtCBF1-Poplar regulonscontained clusters specific for that tissue (Fig 4 gene sub-sets B and C respectively) suggesting different functionalroles for the AtCBF1-Poplar regulons in the annual (leaf)and perennial (stem) tissues However the leaf and stemAtCBF1-Poplar regulons also shared clusters of up-regu-lated genes with each other and with the WT leaf and stemcold regulons (Fig 4 gene subset A and Table 2) Twenty-two of the 63 AtCBF1-Poplar leaf regulon genes identifiedwere also up-regulated in the WT 7 d cold-acclimatedleaves (Table 2) Five of the 26 AtCBF1-Poplar stem regu-lon genes were also up-regulated in WT stems by 7 d coldtreatment (Table 2) Although the total AtCBF1-Poplargene number in stems was lower than that in leavesapproximately the same fraction of the total 7 d coldinduced list could be linked to AtCBF1 expression in thestem (5152 or 3 see Table 2 and Table S3) as in the leaf(22462 or 5 Table 2 and Table S2) Twenty-nine geneswere also found to be down-regulated by AtCBF1 expres-sion in leaves and 24 genes were down-regulated in stems(Table S1c)

Figure 2 Morphology of the AtCBF1-Poplar trees (a) AtCBF1-Poplar were propagated on agar under long-day conditions (b) AtCBF1-Poplar 3 weeks after transfer from propagation on agar to soil (c) AtCBF1-Poplar 10 weeks after transfer from propagation on agar to soil (d) Average plant height during 6 weeks growth on soil in wild-type and AtCBF1-Poplar lines Error bars represent standard error in three independent experiments Open circles wild type closed squares AtCBF1-Poplar line 1 closed triangles AtCBF1-Poplar line 2

Plant age (weeks)

Pla

nthe

ight

(cm

)

0 1 2 3 4 5 60

5

10

15

20

25(d)

(a)

(b)

Line 1 WT

Line 1 WT

(c)

Figure 3 Functional distribution of the genes annotated with known functions significantly up-regulated in warm-grown AtCBF1-Poplar (51 genes in total) and 7 d cold-treated wild-type Populus (387 genes in total) relative to warm-grown wild-type Populus

MetabolismProtein fateDevelopmentCell growth division DNA synthesisTranscriptionEnergyCell rescue defence cell death and ageingTransportation facilitationProtein modificationCellular communication and signal transductionCellular biogenesisStorage

AtCBF1 leaf AtCBF1 stem

WT cold leaf WT cold stem



Comparison of Populus and Arabidopsis CBF regulons

Comprehensive transcriptome changes due to ectopicAtCBF1 expression have previously only been publishedfor Arabidopsis as a combined AtCBF1CBF2CBF3 regu-lon (Fowler amp Thomashow 2002) However paralogue-specific studies have identified gene members of theAtCBF2 (Vogel et al 2005) and AtCBF3 regulons in Ara-bidopsis (Maruyama et al 2004) We next examined the

similarity between our identified annual (leaf) and peren-nial (stem) tissue AtCBF1 regulons in Populus and thereported Arabidopsis CBF regulons We surveyed ourPopulus cDNA microarray DB (Sterky et al 2004) to findall the closest protein homologues to previously reportedCBF-responsive genes from the combined AtCBF1-3 reg-ulon and the AtCBF2- and AtCBF3-paralogue regulonsThe Arabidopsis CBF3 regulon shared the most similaritywith the CBF regulon in Populus Twelve of the 38reported AtCBF3-up-regulated genes that had representa-tive orthologues on our microarray and a gene compari-son showed that more than half (712 having FC gt 13) ofthe AtCBF3-up-regulated gene orthologues present on thePOP1 13K cDNA array were similarly up-regulated in oneor both AtCBF1-Poplar tissues and possessed DREs intheir 1500 bp promoters (Table 3) Examination of theinduction of AtCBF2- and AtCBF123 regulon ortho-logues in Populus (Table S4) showed greater disagree-ment Whether this reduced response of the CBF2 andCBF123 regulon orthologues in Populus to ectopicAtCBF1 expression was indicative of differences in speciesandor paralogue-specific regulon composition remains aquestion that will require additional experiments toanswer

AtCBF1 regulon promoter analysis in Populus reveals conserved linkage of the DRE and abscisic acid-responsive element (ABRE)

Given the broad similarity between the Populus and Ara-bidopsis CBF regulons we determined whether there wasalso a conserved enrichment of the DRE in the promotersof the AtCBF1-Poplar regulon genes Sixteen of the 63(25) AtCBF1-up-regulated leaf regulon gene promoters(defined as the 1500 bp of genomic sequence foundupstream of the ATG site) contained the basic DREnucleic acid sequence lsquoRCCGACrsquo Eight of 26 (31)AtCBF1-up-regulated stem regulon promoters containedthe DRE Positional analysis of the RCCGAC revealed nosignificant enrichment along the 1500 bp promoter How-ever it is worth noting that 7 of 33 DREs appearedbetween 200 and 400 bp upstream of ATG (Table S5)leaving the possibility that a larger sample set using cur-rently unavailable transcriptional start sites instead of theATG would verify the minus450minus51 positional enrichment ofthe DRE reported for Arabidopsis (Maruyama et al 2004)in Populus Recent studies have also noted an enrichmentof the ABRE (Marcotte Russell amp Quatrano 1989)lsquoACGTGTCrsquo in the CBF3 regulon of ArabidopsisMaruyama et al 2004) In Populus an ABRE was found in12 of 87 AtCBF1 regulon 1500 bp promoters representinga significant enrichment (χ2 P lt 005) There was also asignificant overlap between ABRE- and DRE-containinggene promoters in Populus (412 ABRE-containing genepromoters also contained the RCCGAC consensus χ2P lt 0001) and positional enrichment of the ABRE at posi-tions 100ndash199 bp and 300ndash399 bp upstream of the startcodon (χ2 P lt 0001)

Figure 4 Hierarchical tree of genes belonging to the CBF regulon based on similarities in expression profiles of the leaf and stem tissues of AtCBF1-Poplar and 7 d cold-treated wild-type Populus Gene subsets up-regulated in the leaf and stem tissue of AtCBF1-Poplar are indicated to the right with brackets where (A) indicates the gene subset similarly up-regulated in both AtCBF1-Poplar and wild-type cold-treated Populus tissues (B) indicates the AtCBF1-Poplar leaf-specific regulon and (C) indicates the AtCBF1-Poplar stem-specific regulon

50

30

20

12

10

08

06

04

Rel

ativ

eex

pres

sion

(fol

d-ch

ange

)

AtCBF1 WT Cold

L SSL

A

B

C



The Populus balsamifera ssp trichocarpa genome encodes six CBF-like transcription factorsThe AtCBF1-Poplar leaf and stem regulons containedmany genes previously linked to the process of cold accli-

mation in herbaceous annuals such as Arabidopsis andwhen combined with the observed improvement in FT inAtCBF1-Poplar implied that cold-responsive CBF tran-scription factors were encoded by the Populus genome andinvolved in the cold acclimation process Iterative

Table 2 A selection of genes up-regulated in AtCBF1-Poplar (Poplar tremula times alba) and wild-type (WT) Populus tremula times alba cold acclimated for 1 week at 2 degC (versus WT Populus tremula times alba grown at 23 degC) Normalized mean fold-change (FC) values are shown (bold face indicates P lt 005) The poplar gene probe (PU) number is matched to its closest Arabidopsis gene (AGI) based on homology to the Arabidopsis genome sequence This table corresponds to gene subset lsquoArsquo in Fig 4

CBF1Leaf FC

CBF1Stem FC

WTLeaf FC

WT Stem FC Clone ID AGI Description Category

21 11 37 28 PU03647 At1g54410 SRC2 Cell rescue20 10 50 43 PU03776 At5g66780 Expressed protein Unknown role22 20 35 34 PU04037 At2g21120 P0491F1121 protein Oryza sativa Metabolism18 15 31 22 PU03803 At2g19370 Expressed protein Unknown role20 14 50 27 PU03505 At5g09390 Expressed protein Unknown role22 20 123 52 PU03995 At3g06660 Expressed protein Unknown role20 19 79 37 PU01895 At2g15970 WCOR413-like protein Cell rescue18 20 78 34 PU03971 At4g39450 Expressed protein Unknown role19 19 100 58 PU03418 At3g05880 LTI6A Cell rescue26 24 145 41 PU03755 At5g38760 Pollen coat protein Development21 17 94 32 PU03503 None Arabinogalactan protein AGP21 Development25 13 25 28 PU03276 At5g61660 Glycine-rich protein Unknown role28 11 35 22 PU03151 At1g71691 GDSL-motif lipasehydrolase Metabolism25 13 94 13 PU03398 At4g29680 Nucleotide pyrophosphatase Metabolism26 25 140 13 PU03390 At2g28680 Legumin-like protein Storage18 16 85 16 PU03329 At5g67080 Expressed protein Unknown role22 18 74 15 PU00815 At4g24220 Dehydrin Cell rescue21 15 47 25 PU03208 At5g54470 CONSTANS B-box zinc finger protein Transcription19 11 23 minus13 PU10341 At5g55430 Expressed protein Unknown role18 minus17 23 20 PU00410 None Expressed protein Unknown role10 21 13 40 PU10603 At2g41380 Embryo abundant protein Unknown role10 18 12 18 PU10452 At5g39670 Calcium binding protein Signal transduction10 18 17 16 PU03954 At5g62165 MADS-box transcription factor FBP22 Transcription11 20 46 48 PU03748 At1g06330 Copper-binding protein Unknown role

Table 3 Normalized microarray expression values for Populus genes matched to reported Arabidopsis CBF3 regulon members in AtCBF1-Poplar and trees acclimated for 1 week at 2 degC The poplar gene probe (PU) number is matched to its closest Arabidopsis gene (AGI) based on homology to the Arabidopsis genome sequence Gene names presented in bold have a dehydration-responsive element (DRE) cis-element in within 1500 bp of the start codon

AtCBF1 trees WT cold trees

Leaf FC Stem FC Leaf FC Stem FC

CBF3 regulonName CloneID AGIPdc1 PU00414 At4g33070 11 12 14 10ATFP6 PU00465 At4g38580 13 12 75 29expressed protein PU07867 At4g35300 11 10 20 13expressed protein PU04708 At5g62530 15 11 25 17expressed protein PU10962 At4g14000 11 10 12 11Lti6 PU10681 At4g30650 11 09 52 13erd7 PU03488 At2g17840 13 15 25 28expressed protein PU11420 At1g27730 12 12 15 14expressed protein PU07579 At1g51090 12 14 10 13rap21 PU10438 At1g46768 14 17 14 18expressed protein PU05694 At4g15910 15 09 15 08src2 PU10619 At1g54410 15 06 12 13

WT wild type FC fold-change



Figure 5 Comparison of representative C-repeat binding factor (CBF) protein family members in dicots (a) Alignment of AP2 and flanking region of CBF factors (b) Phylogenetic analysis based on minimum evolution was performed with the full-length protein sequences (Fig S1) Polypeptides used for alignments were the six CBFs of Arabidopsis AtCBF1 (U77378) AtCBF2 (AF074601) AtCBF3 (AF074602) AtCBF4 (NM_124578) AtDDF1At1g12610 (NM_101131) AtDDF2At1g63030 and (NM_104981) three Brassica napus CBFs BnCBF-A (AAL38242) BnCBF-B (AAL38243) BnCBF-C (AAD45623) two tobacco EREBPs Nt111A (AAG43548) Nt111B (AAG43549) Prunus avium PaDREB1 (AB121674) and the six poplar CBFs identified in this study PtCBF1 (JGI666968) PtCBF2 (JGI346104) PtCBF3 (JGI548519) PtCBF4 (JGI63608) PtCBFL1 (JGI594822) and PtCBFL2 (JGI649103) PtDRTY (JGI253714) was also used and is a related AP2 domain protein with extended homology to CBFs in the C-terminal domain Arabidopsis TINY (AtTINY) (AAC29139) was utilised as an outlier and represents the closest AP2 domain-containing protein group that falls outside the CBFDREB1 family clade A likely poplar TINY orthologue PtTINY-Like (JGI653993) was also utilised as an outlier and assembled from the draft poplar genome sequence data CBF proteins were initially aligned using CLUSTAL-W the output alignments hand refined using GeneDoc Version 26 software (httpwwwpscedubiomedgenedoc) and used to conduct phylogenetic analysis using MEGA Version 21 software (httpwwwmegasoftwarenet) Phylogenetic trees were generated using MEGA Version 21 Neighbor Joining and Minimum Evolution methodologies on 1000 bootstrap replications Bootstrap numbers are shown on the dendrogram branch points

(a)

PtCBF1 PKKRAGRKKFRETRHPVYRGVRRRNSGKWVCEVREPNKKS-RIWLGTFPTADMAARAHDVAALALRGRSACLNFADSAWRLPtCBF2 PKKRAGRKKFRETRHPVYRGVRRRNSGKWVCEVREPNKKS-RIWLGTFPTAEMAARAHDVAALALRGRSACLNFADSAWRLNt111B PKKRAGRKKFRETRHPVYRGVRKRNSDKWVCELREPNKKS-RIWLGTFPSAEMAARAHDVAAIALRGRSACLNFADSAWKLNt111A PKKRAGRKKLRETRHPVYRGVRKRNSGKWVCEVREPNKQS-RIWLGTFPSAEMAVRAHDVAAIALRGRSACLNFADSAWKLAtCBF2 PKKPAGRKKFRETRHPIYRGVRQRNSGKWVCELREPNKKT-RIWLGTFQTAEMAARAHDVAAIALRGRSACLNFADSAWRLAtCBF3 PKKPAGRKKFRETRHPIYRGVRRRNSGKWVCEVREPNKKT-RIWLGTFQTAEMAARAHDVAALALRGRSACLNFADSAWRLAtCBF1 PKKPAGRKKFRETRHPIYRGVRQRNSGKWVSEVREPNKKT-RIWLGTFQTAEMAARAHDVAALALRGRSACLNFADSAWRLBnCBF-A PKKPAGRKKFRETRHPIYRGVRLRKSGKWVCEVREPNKKS-RIWLGTFKTAEIAARAHDVAALALRGRGACLNFADSAWRLBnCBF-B PKKPAGRKKFQETRHPIYRGVRLRKSGKWVCEVREPNKKS-RIWLGTFKTAEIAARAHDVAALALRGRGACLNFADSAWRLBnCBF-C PKKPAGRKKFRETRHPVYRGVRLRNSGKWVCEVREPNKKS-RIWLGTFLTAEIAARAHDVAAIALRGKSACLNFADSAWRLAtCBF4 PKKRAGRKKFRETRHPIYRGVRQRNSGKWVCEVREPNKKS-RIWLGTFPTVEMAARAHDVAALALRGRSACLNFADSAWRLPtCBF3 PKKRAGRRIFKETRHPIFRGVRKRNGDKWVCELREPNKKS-RIWLGTYPTPEMAARAHDVAALAFRGKSACLNFADSAWRLPtCBF4 PKKRAGRRIFRETRHPVFRGVRKRNGNKWVCEMREPNKKS-RIWLGTYPTPEMAARAHDVAALALRGKSACLNFADSAWRLPaDREB1 PKKRAGRRVFKETRHPVYRGVRRRNNDKWVCEMREPNKKKSRIWLGTYPTAEMAARAHDVAALAFRGKLACINFADSAWRLAtDdf2 PKKRAGRRIFKETRHPIYRGVRRRDGDKWVCEVREPIHQR-RVWLGTYPTADMAARAHDVAVLALRGRSACLNFSDSAWRLAtDdf1 PKKRAGRRVFKETRHPVYRGIRRRNGDKWVCEVREPTHQR-RIWLGTYPTADMAARAHDVAVLALRGRSACLNFADSAWRLPtCBFL1 KKKKAGRKKFKETRHPVYRGVRKRNGNKWVCEVREPNKKS-RIWLGTFTSPEMAARAHDVAALALKGETATLNFPDSALILPtCBFL2 KKNKAGRKKFKETRHPVYRGVRRRNGNKWVCEVREPNKKS-RIWVGTFKSPEMAARAHDVAALALKGELAALNFLDSALILPt-DRTY RPPGGSSAQPASGRHPTFKGVRLR-SGKWVSEIREPRKTT-RVWLGTYPTPEMAATAYDVAALALKGTNTPLNFPESILSYPtTINY -PCKKITRIRDSSKHPVYRGVRMRNWGKWVSEIREPRKKS-RIWLGTFPTPEKAARAHDAAALSIKGNSAILNFPELANSLAtTINY --------VKDSGKHPVYRGVRKRNWGKWVSEIREPRKKS-RIWLGTFPSPEMAARAHDVAALSIKGASAILNFPDLAGSF

AP2 domain

AtCBF2

AtCBF3

AtCBF1

BnCBF-C

BnCBF-A

BnCBF-B

AtCBF4

PtCBF1

PtCBF2

Nt111B

Nt111A

PtCBF3

PtCBF4

PaDREB1

AtDdf2

AtDdf1

PtCBFL1

PtCBFL2

PtDRTY

PtTINY-Like

AtTINY100

100

100

99

99

100

94

100

56

92

91 75

97

100 65

82

8663

(b)



tBLASTn searches of the Populus balsamifera ssp tri-chocarpa (Torr amp A Gray) Brayshaw genome (httpgenomejgi-psforgPoptr1Poptr1homehtml) using theconserved AP2 and flanking CBF ID sequences from Ara-bidopsis (Jaglo et al 2001) identified six potential CBF-encoding genes An amino acid sequence alignment of thehighly conserved AP2 domain and flanking CBF ID regionsof the candidate poplar CBFs with other dicotyledonousCBFs (Fig 5a) shows that two of the six candidate genes(PtCBF1 and PtCBF2) have 100 conservation of the pre-viously reported Arabidopsis CBF consensus sequences(PKKRPRAGRxKFxETRHP and DSAWR) two othercandidate genes (PtCBF3 and PtCBF4) possess a singleamino acid substitution in the N-terminal consensus (Iinstead of K in position 10) with the remaining twosequences (PtCBFL1 and PtCBFL2) deviating from theconsensus sequences by four and five amino acids respec-tively Interestingly none of the poplar CBFs utilizes thecommonly occurring proline at the fourth position butPtCBF1-4 use the common alternate arginine residue whilePtCBFL1-2 have lysine at this position in contrast to allthe other dicot CBFs A phylogenetic analysis of the sixpoplar CBF candidates with known full-length sequencesfrom dicotyledonous plants using the TINY AP2 transcrip-tion factor as the out-group confirms that PtCBFL1 andPtCBFL2 share the least amino acid similarity to the char-acterized CBFs from Arabidopsis (Fig 5b) Also of somenote a Populus gene previously reported to be lsquoCBF1-likersquoand up-regulated in dormant cambium (PtDRTY)(Schrader et al 2004) groups with TINY not the CBFsHowever unlike TINY PtDRTY shares some extendedblocks of homology in the C-terminal domain with theCBFs Investigation of the 1500 bp promoters of thePtCBFs also demonstrated that they possess numerouspotential ICE-binding sites (CANNTG) and a tBLASTnsearch of the Populus balsamifera ssp trichocarpa genome

identified Populus-encoded ICE proteins with high similar-ity to Arabidopsis ICE1 (data not shown) indicating thatICE-mediation of the CBF signalling pathway may also beconserved in this woody perennial

PtCBFs are cold-inducible in leaf and stem tissues

Based on the phylogenetic analysis we selected PtCBF1-4for further study and real-time PCR demonstrated that allwere cold-inducible although the kinetics of induction andtissue specificity differed between the orthologues (Fig 6)PtCBF1 was cold-inducible in leaf and stem tissue PtCBF1transcript levels in both tissue types peaked 6 h after trans-fer to cold and decreased again overnight (Fig 6a) Simi-larly PtCBF3 was inducible in both leaf and stem tissue(Fig 6c) PtCBF3 transcript accumulated rapidly after cold-shifting peaking at 3 h and returned to near starting levelswithin 6 h In contrast both PtCBF2 and PtCBF4 werecold-inducible in leaf tissue but were only weakly cold-induced in the stem (Fig 6b amp d) Leaf PtCBF2 expressionpeaked 9 h after transfer with stem transcripts peakingbetween 3 and 6 h PtCBF4 expression peaked at 3 h(Fig 6d) These data strongly support the conclusion thatpoplar utilizes a cold-inducible CBF-based response tocold

DISCUSSION

In this study we have characterised the role of the CBFtranscription factors in leaves and stems of a woody peren-nial Populus As shown previously for herbaceous plants(Jaglo et al 2001 Hsieh et al 2002 Lee et al 2003 2004)ectopic expression of AtCBF1 from Arabidopsis increasedthe constitutive FT of non-acclimated meristematic stemand leaf tissues in both independent AtCBF1-Poplar lines

Figure 6 Expression response of Populus CBF paralogues to low temperature in wild-type trees (a) PtCBF1 (b) PtCBF2 (c) PtCBF3 (d) PtCBF4 Expression was determined by RT-PCR Each reaction used 30 ng first-strand cDNA and normalized to 18S rRNA quantity Open circle leaf closed circle stem Error bars represent the standard error on three independent experiments

Rel

ativ

eab

unda

nce

Time (h)after transfer to 5 degC

0 4 8 12 16 20 24

0

2

4

6StemLeaf

0

2

4

6PtCBF1(a) (b)

(c) (d)

0

2

4

6PtCBF3

PtCBF2

PtCBF4

Relative

abundance

0 4 8 12 16 20 2400

02

04

06

08

10

12

14



demonstrating that the CBF-mediated signalling pathwayand regulon are conserved in annual and perennial tissuesof woody perennials This conclusion is supported by earlierreports that the promoters of cold-responsive genes such asBplti36 from birch are activated by Arabidopsis CBFs(Puhakainen et al 2004) and that CBF orthologues fromwoody species are functional when transferred to Arabi-dopsis (Kitashiba et al 2004) Our subsequent microarrayanalysis showing broad agreement between the transcrip-tomes of AtCBF1-Poplar and that reported for theAtCBF1-3 regulon orthologues in Arabidopsis (Fowler ampThomashow 2002 Vogel et al 2005) demonstrates thatAtCBF1 acts in a similar manner in Populus as AtCBF1-3act in Arabidopsis

Interestingly the functional distribution of the PopulusAtCBF1 regulon genes differed between the annual (leaf)and perennial (stem) tissues of this woody species In stemtissues 59 of the AtCBF1-Poplar regulon was composedof genes involved in the processes of lsquoTranscriptionrsquo andlsquoCellular CommunicationSignal Transductionrsquo while thesetwo processes composed only 26 of the AtCBF1-Poplarleaf regulon It has been shown previously that the CBFregulon contains a number of transcription factors whichthemselves target additional gene sets (Fowler amp Tho-mashow 2002 Gilmour et al 2004 Novillo et al 2004Vogel et al 2005 Nakashima amp Yamaguchi-Shinozaki2006) However the difference in composition of the pop-lar leaf and stem regulons was unexpected and could notbe attributed to a general difference in the low temperaturetranscriptomes of the annual and perennial tissues becausethe same comparison made between 7 d cold-acclimatedWT leaf and stem tissues demonstrated general agreementin the distribution of up-regulated functional categories(Fig 3) Examination of the identities of the leaf and stemcold regulon genes showed that their functional similaritieswere not superficial the WT cold-responsive regulons ofthe leaf and stem were compositionally similar It is possi-ble that the AtCBF1-Poplar leafstem regulon functionaldifference is due to the small size of the stem regulonhowever examination of the largest functional group(lsquoMetabolismrsquo) in the leaf and stem regulons contained sig-nificantly (χ2 P lt 005) fewer genes (2) than expected (6)in the stem regulon The composition of the AtCBF1-Pop-lar stem regulon was also not representative of the func-tional distribution of the genes printed on our arrayindicating that this compositional difference has a biologi-cal basis

Associated with the difference in regulon compositionbetween annual leaf tissue and the perennial stem meristemtissue another finding was that a FRY2 orthologue wasdown-regulated in AtCBF1-Poplar leaves FRY2 is a dou-ble stranded RNA-binding transcriptional repressor that isknown to repress expression of CBF regulon members byworking at a point upstream of their transcription (Xionget al 2002) Like the majority of genes down-regulated inthe AtCBF1-Poplar the FRY2 1500 bp promoter lacks theDRE consensus indicating that a CBF-responsive tran-scription factor or post-transcriptional process and not

CBF itself leads to its differential expression Neverthelessbecause the fry2 mutation is known to alter ABA sensitivityin Arabidopsis seeds and seedlings (Xiong et al 2002) andthe CBF regulon in Populus was significantly enriched forthe ABRE differential expression of FRY2 in leaves (ver-sus stems) of AtCBF1-Poplar may also have contributed tothe CBF leafstem regulon differences The up-regulationof PtCBF3 in AtCBF1-Poplar stem tissues also suggeststhat individual Populus CBF factors are as in Arabidopsis(Novillo et al 2004) regulated by their own paraloguesPtCBF promoters contain numerous ICE1 binding sitesand the genome encodes ICE1-like proteins (data notshown)

Despite the broad similarity between the Populus andArabidopsis CBF leaf regulons and the substantial overlapof the AtCBF1-Poplar leaf regulon with the endogenoustranscriptional response to low temperature we found nosignificant enrichment of the DRE (RCCGAC) in the1500 bp promoters of our identified Populus CBF regulonThe lack of DRE enrichment may have been due tothe small number of genes identified in the regulon but thecurrent lack of annotated transcriptional start sites in thePopulus genome assembly the larger genome and inter-genic size of Populus or an altered AtCBF1-binding con-sensus sequence may all have contributed to this Incontrast the ABRE previously shown to be enriched inCBF3 regulon promoters and to impart ABA-responsive-ness (Hattori et al 2002) showed significant positionalenrichment (between 100 and 400 bp upstream of the ATG)and co-occurrence with the DRE in our Populus CBFregulon indicating that this cis-element relationship isimportant

As noted earlier the accumulation of CBF transcriptsand the activity of the CRT regulatory motif in Arabidopsisare also modulated by the presence and quality of lightduring cold stress (Kim et al 2002 Fowler et al 2005) andthat CRT-driven GUS reporter activity is altered in phyto-chrome mutant backgrounds suggesting that day lengthmediates expression of the CBF regulon through phyto-chrome B in Arabidopsis (Kim et al 2002) Pre-treatmentwith short days has also been shown to enhance the expres-sion of low temperature-responsive CRT-containing genesin birch (Puhakainen et al 2004) and a CBF-like transcrip-tion factor is induced by short days in the cambial meristemof poplar (Schrader et al 2004) These data suggest thatCBF or related transcriptional regulators may be involvedin the day-length-mediated growth cessation in woody spe-cies ABA is known to mediate the response of many abi-otic stress-response genes (Chen Li amp Brenner 1983Maumlntyla Laringng amp Palva 1995) via activation of the ABRE(Marcotte et al 1989 Lam amp Chua 1991) and initiation andmaintenance of bud dormancy in woody species has beenassociated with high endogenous ABA levels (Wright 1975Rinne Saarelainen amp Junttila 1994 Rinne Tuominen ampJunttila 1994 Rohde et al 2002 Li et al 2003b) The strongconcurrence of the DRE and ABRE in the promoters ofour Populus CBF regulon supports the conclusion thatthere is direct interaction between the day-length-



dependent ABA and low-temperature-dependent CBF sig-nalling pathways Although unravelling the role(s) that thedifferent PtCBFs play in this interaction will require addi-tional studies our data nevertheless indicate that in poplarthe initial perception of low temperature (biologically indi-cated by the accumulation of CBF transcripts in WT treesand artificially mimicked by ectopic expression of AtCBF1transcript in AtCBF1-Poplar) results in up-regulation of afunctionally different CBF regulon in annual leaf andperennial stem tissues This conclusion is supported by thedifferential inducibility of the endogenous PtCBFs in Pop-ulus Although all four PtCBFs are cold-inducible in leaftissue only PtCBF1 and PtCBF3 are significantly inducedin the stem PtCBF12 and PtCBF34 form distinct phylo-genetic clusters and one gene from each cluster is tissue-specific suggesting that the different CBF gene clustersmay be controlling slightly different CBF regulons in thedifferent tissues In accordance with their predicted rolein cold acclimation in trees (protection of temperature-sensitive tissues such as buds and flowers during springflushing) the poplar expressed sequence tag (EST) sequenc-ing project (Sterky et al 2004) has also detected PtCBF3expression in flower buds (as well as roots) and PtCBF1in dormant buds

Overall our studies of the CBF regulon in Populus havedemonstrated that the central role played by the CBF familyof transcriptional activators in cold acclimation of herba-ceous annuals such as Arabidopsis has been maintained intemperate woody perennials despite the evolutionary pres-sure imposed by the development of a winter dormant phe-notype However these results also point to several keydifferences when we compare results from Populus withArabidopsis (1) unlike data from all herbaceous speciestwo of the four poplar CBF paralogues show differentialexpression in leaf (annual) and stem (perennial) tissues (2)the functional composition of the stem and leaf AtCBF1-Poplar regulons are different perhaps reflecting in part thedifferent transcriptomes of these divergent tissues and alsopossibly indicating that different functional requirements inthese tissues have driven the evolution of specific leaf andstem cold-responsive CBF paralogues and (3) this diver-gence in functional composition is reinforced by the findingof leaf- and stem-specific AtCBF1-Poplar regulon membersTaken together these data suggest that the perennial-driven evolution of winter dormancy has led to thedevelopment of specific roles for abiotic stress responseregulators such as the CBFs in annual and perennial tissuesFuture studies exploring the FT and transcriptomic changesin poplar constitutivelyinducibly expressing native PtCBFsshould help elucidate these specific roles in greater detail

ACKNOWLEDGMENTS

We thank Joel Davis for excellent technical assistance Wealso thank Dr Mike Thomashow for providing the originalAtCBF1 expression cassette Zoran Jeknic for providingthe plasmid construct pGAH35SAtCBF1 used in this studyand Andreas Sjoumldin for assistance with programming the

normalization protocol in lsquoRrsquo This study was supported inpart by grants from the Swedish Forestry and AgriculturalResearch Council to VH from Statens Energimyndigheten(STEM) to RB from Natural Sciences and EngineeringResearch Council (NSERC) to NPAH and from theNational Science Foundation (NSF) Plant Genome Project(DBI 0110124) to THHC

REFERENCES

Andersson A Keskitalo J Sjodin A et al (2004) A transcrip-tional timetable of autumn senescence Genome Biology 5 R24

Arora R amp Wisniewski M (1994) Cold acclimation in geneticallyrelated (sibling) deciduous and evergreen peach (Prunus persica(L) Batsch) a 60-kilodalton bark protein in cold-acclimatedtissues of peach is heat stable and related to the dehydrin familyof proteins Plant Physiology 105 95ndash101

Arora R Wisniewski M amp Rowland LJ (1996) Cold acclimationand alterations in dehydrin-like and bark storage proteins in theleaves of sibling deciduous and evergreen peach Journal of theAmerican Society for Horticultural Science 121 915ndash919

Bravo LA Gallardo J Navarrete A Olave N Martinez JAlberdi M Close TJ amp Corcuera LJ (2003) Cryoprotectiveactivity of a cold-induced dehydrin purified from barley Physi-ologia Plantarum 118 262ndash269

van Buskirk HA amp Thomashow MF (2006) Arabidopsis tran-scription factors regulating cold acclimation Physiologia Plan-tarum 126 72ndash80

Campalans A Pages M amp Messeguer R (2000) Protein analysisduring almond embryo development identification and charac-terization of a late embryogenesis abundant protein Plant Phys-iology and Biochemistry 38 449ndash457

Chen HH Li PH amp Brenner ML (1983) Involvement of absci-sic acid in potato cold acclimation Plant Physiology 71 362ndash365

Christersson L (1978) The influence of photoperiod and temper-ature on the development of frost hardiness in seedlings ofPinus sylvestris and Picea abies Physiologia Plantarum 44288ndash294

DeBlock M (1990) Factors influencing the tissue culture and theAgrobacterium tumefaciens-mediated transformation of hybridaspen and poplar clones Plant Physiology 93 1110ndash1116

Dexter ST Tottingham WE amp Garber LG (1932) Investigationof hardiness of plants by measurement of electrical conductivityPlant Physiology 7 63ndash78

Fowler S amp Thomashow MF (2002) Arabidopsis transcriptomeprofiling indicates that multiple regulatory pathways are acti-vated during cold acclimation in addition to the CBF coldresponse pathway Plant Cell 14 1675ndash1690

Fowler SG Cook D amp Thomashow MF (2005) Low tempera-ture induction of Arabidopsis CBF1 2 and 3 is gated by thecircadian clock Plant Physiology 137 961ndash968

Fuchigami LH Weiser CJ amp Evert DR (1971) Induction of coldacclimation in Cornus stolonifera Michx Plant Physiology 4798ndash103

Gilmour SJ Sebolt AM Salazar MP Everard JD amp Tho-mashow MF (2000) Overexpression of the Arabidopsis CBF3transcriptional activator mimics multiple biochemical changesassociated with cold acclimation Plant Physiology 124 1854ndash1865

Gilmour SJ Fowler SG amp Thomashow MF (2004) Arabidopsistranscriptional activators CBF1 CBF2 and CBF3 have match-ing functional activities Plant Molecular Biology 54 767ndash781

Haake V Cook D Riechmann JL Pineda O Thomashow MFamp Zhang JZ (2002) Transcription factor CBF4 is a regulator of



drought adaptation in Arabidopsis Plant Physiology 130 639ndash648

Hattori T Totsuka M Hobo T Kagaya Y amp Yamamoto-ToyodaA (2002) Experimentally determined sequence requirement ofACGT-containing abscisic acid response element Plant and CellPhysiology 43 136ndash140

Hertzberg M Sievertzon M Aspeborg H Nilsson P SandbergG amp Lundeberg J (2001) cDNA microarray analysis of smallplant tissue samples using a cDNA tag target amplification pro-tocol Plant Journal 25 585ndash591

Howell GS amp Stockhause SS (1973) The effect of defoliationtime on acclimation and dehardening in tart cherry (Prunuscerasus L) Journal of the American Society for HorticulturalScience 98 132ndash136

Hsieh TH Lee JT Chang YY amp Chan MT (2002) Tomatoplants ectopically expressing Arabidopsis CBF1 show enhancedresistance to water deficit stress Plant Physiology 130 618ndash626

Hughes DW amp Galau GA (1988) Preparation of RNA fromcotton leaves and pollen Plant Molecular Biology Reporter 6253ndash275

Jaglo KR Kleff S Amundsen KL Zhang X Haake V ZhangJZ Deits T amp Thomashow MF (2001) Components of theArabidopsis C-repeatdehydration-responsive element bindingfactor cold-response pathway are conserved in Brassica napusand other plant species Plant Physiology 127 910ndash917

Jaglo-Ottosen KR Gilmour SJ Zarka DG Schabenberger Oamp Thomashow MF (1998) Arabidopsis CBF1 overexpressioninduces COR genes and enhances freezing tolerance Science280 104ndash106

Junttila O (1976) Apical growth cessation and shoot tip abscissionin Salix Physiologia Plantarum 38 278ndash286

Kim HJ Kim YK Park JY amp Kim J (2002) Light signallingmediated by phytochrome plays an important role in cold-induced gene expression through the C-repeatdehydrationresponsive element (CDRE) in Arabidopsis thaliana PlantJournal 29 693ndash704

Kitashiba H Matsuda N Ishizaka T Nakano H amp Suzuki T(2002) Isolation of genes similar to DREB1CBF from sweetcherry (Prunus avium L) Journal of the Japanese Society forHorticultural Science 71 651ndash657

Kitashiba H Ishizaka T Isuzugawa K Nishimura K amp SuzukiT (2004) Expression of a sweet cherry DREB1CBF orthologin Arabidopsis confers salt and freezing tolerance Journal ofPlant Physiology 161 1171ndash1176

Lam E amp Chua NH (1991) Tetramer of a 21-base pair syntheticelement confers seed expression and transcriptional enhance-ment in response to water-stress and abscisic-acid Journal ofBiological Chemistry 266 17131ndash17135

Lee JT Prasad V Yang PT Wu JF Ho THD Chang YYamp Chan MT (2003) Expression of Arabidopsis CBF1 regulatedby an ABAstress inducible promoter in transgenic tomato con-fers stress tolerance without affecting yield Plant Cell amp Envi-ronment 26 1181ndash1190

Lee SC Huh KW An K An G amp Kim SR (2004) Ectopicexpression of a cold-inducible transcription factor CBF1DREB1b in transgenic rice (Oryza sativa L) Molecules andCells 18 107ndash114

Li C Puhakainen T Welling A Vihera-Aarnio A Ernstsen AJunttila O Heino P amp Palva ET (2002) Cold acclimation insilver birch (Betula pendula) Development of freezing tolerancein different tissues and climatic ecotypes Physiologia Plantarum116 478ndash488

Li CY Junttila O Ernstsen A Heino P amp Palva ET (2003a)Photoperiodic control of growth cold acclimation and dor-mancy development in silver birch (Betula pendula) ecotypesPhysiologia Plantarum 117 206ndash212

Li CY Vihera-Aarnio A Puhakainen T Junttila O Heino Pamp Palva ET (2003b) Ecotype-dependent control of growthdormancy and freezing tolerance under seasonal changes in Bet-ula pendula Roth Trees ndash Structure and Function 17 127ndash132

Lim CC Krebs SL amp Arora R (1999) 25-kDa dehydrin associ-ated with genotype and age-dependent leaf freezing-tolerancein Rhododendron a genetic marker for cold hardiness Theo-retical and Applied Genetics 99 912ndash920

Maumlntyla E Laringng V amp Palva ET (1995) Role of abscisic-acid indrought-induced freezing tolerance cold acclimation and accu-mulation of LTI78 and RAB18 proteins in Arabidopsis thalianaPlant Physiology 107 141ndash148

Marcotte WR Russell SH amp Quatrano RS (1989) Abscisicacid-responsive sequences from the Em gene of wheat PlantCell 1 969ndash976

Maruyama K Sakuma Y Kasuga M Ito Y Seki M Goda HShimada Y Yoshida S Shinozaki K amp Yamaguchi-ShinozakiK (2004) Identification of cold-inducible downstream genes ofthe Arabidopsis DREB1ACBF3 transcriptional factor usingtwo microarray systems Plant Journal 38 982ndash993

Nakashima K amp Yamaguchi-Shinozaki K (2006) Regulonsinvolved in osmotic stress-responsive and cold stress-responsivegene expression in plants Physiologia Plantarum 126 62ndash71

Novillo F Alonso JM Ecker JR amp Salinas J (2004) CBF2DREB1C is a negative regulator of CBF1DREB1B and CBF3DREB1A expression and plays a central role in stress tolerancein Arabidopsis Proceedings of the National Academy of Scienceof the USA 101 3985ndash3990

Onouchi H Yokoi K Machida C Matsuzaki H Oshima YMatsuoka K Nakamura K amp Machida Y (1991) Operation ofan efficient site-specific recombination system of Zygosaccharo-myces rouxii in tobacco cells Nucleic Acids Research 19 6373ndash6378

Owens CL Thomashow MF Hancock JF amp Iezzoni AF(2002) CBF1 orthologs in sour cherry and strawberry and theheterologous expression of CBF1 in strawberry Journal of theAmerican Society for Horticultural Science 127 489ndash494

Puhakainen T Li C Boije-Malm M Kangasjarvi J Heino P ampPalva ET (2004) Short-day potentiation of low temperature-induced gene expression of a C-repeat-binding factor-controlledgene during cold acclimation in silver birch Plant Physiology136 4299ndash4307

Richard S Morency MJ Drevet C Jouanin L amp Seguin A(2000) Isolation and characterization of a dehydrin gene fromwhite spruce induced upon wounding drought and cold stressesPlant Molecular Biology 43 1ndash10

Rinne P Saarelainen A amp Junttila O (1994) Growth cessationand bud dormancy in relation to ABA level in seedlings andcoppice shoots of Betula pubescens as affected by a short pho-toperiod Physiologia Plantarum 90 451ndash458

Rinne P Tuominen H amp Junttila O (1994) Seasonal changes inbud dormancy in relation to bud morphology water and starchcontent and abscisic acid concentration in adult trees of Betulapubescens Tree Physiology 14 549ndash561

Rinne P Welling A amp Kaikuranta P (1998) Onset of freezingtolerance in birch (Betula pubescens Ehrh) involves LEA pro-teins and osmoregulation and is impaired in an ABA-deficientgenotype Plant Cell amp Environment 21 601ndash611

Rohde A Prinsen E De Rycke R Engler G Van Montagu Mamp Boerjan W (2002) PtABI3 impinges on the growth and dif-ferentiation of embryonic leaves during bud set in poplar PlantCell 14 1885ndash1901

Sakuma Y Liu Q Dubouzet JG Abe H Shinozaki K ampYamaguchi-Shinozaki K (2002) DNA-binding specificity of theERFAP2 domain of Arabidopsis DREBs transcription factorsinvolved in dehydration- and cold-inducible gene expression



Biochemical and Biophysical Research Communications 290998ndash1009

Saxe H Cannell MGR Johnsen O Ryan MG amp Vourlitis G(2001) Tree and forest functioning in response to global warm-ing New Phytologist 149 369ndash400

Schrader J Moyle R Bhalerao R Hertzberg M Lundeberg JNilsson P amp Bhalerao RP (2004) Cambial meristem dormancyin trees involves extensive remodelling of the transcriptomePlant Journal 40 173ndash187

Selas V Piovesan G Adams JM amp Bernabei M (2002) Climaticfactors controlling reproduction and growth of Norway sprucein southern Norway Canadian Journal of Forest Research 32217ndash225

Sterky F Bhalerao RR Unneberg P et al (2004) A PopulusEST resource for plant functional genomics Proceedings of theNational Academy of Science of the USA 101 13951ndash13956

Stockinger EJ Gilmour SJ amp Thomashow MF (1997) Arabi-dopsis thaliana CBF1 encodes an AP2 domain-containing tran-scriptional activator that binds to the C-repeatDRE a cis-actingDNA regulatory element that stimulates transcription inresponse to low temperature and water deficit Proceedings ofthe National Academy of Science of the USA 94 1035ndash1040

Sukumaran NP amp Weiser CJ (1972) An excised leaflet test forevaluating potato frost tolerance Hortscience 7 467ndash468

Svensson J Ismail AM Palva ET amp Close TJ (2002) Dehy-drins In Sensing Signaling and Cell Adaptation (eds KB Storeyamp JM Storey) pp 155ndash171 Elsevier Amsterdam the Nether-lands

Vogel JT Zarka DG Van Buskirk HA Fowler SG amp Tho-mashow MF (2005) Roles of the CBF2 and ZAT12 transcrip-tion factors in configuring the low temperature transcriptome ofArabidopsis Plant Journal 41 195ndash211

Weiser CJ (1970) Cold resistance and injury in woody plantsScience 169 1269ndash1278

Welling A Kaikuranta P amp Rinne P (1997) Photoperiodic induc-tion of dormancy and freezing tolerance in Betula pubescensinvolvement of ABA and dehydrins Physiologia Plantarum 100119ndash125

Welling A Moritz T Palva ET amp Junttila O (2002) Indepen-dent activation of cold acclimation by low temperature andshort photoperiod in hybrid aspen Plant Physiology 1291633ndash1641

Wisniewski M Close TJ Artlip T amp Arora R (1996) Seasonalpatterns of dehydrins and 70-kDa heat-shock proteins in barktissues of eight species of woody plants Physiologia Plantarum96 496ndash505

Wisniewski M Webb R Balsamo R Close TJ Yu XM ampGriffith M (1999) Purification immunolocalization cryoprotec-tive and antifreeze activity of PCA60 a dehydrin from peach(Prunus persica) Physiologia Plantarum 105 600ndash608

Wright STC (1975) Seasonal changes in the levels of free andbound abscisic acid in blackcurrent (Ribes nigrum) buds and

beech (Fagus sylvatica) buds Journal of Experimental Botany26 161ndash174

Xiong LM Lee H Ishitani M Tanaka Y Stevenson B KoiwaH Bressan RA Hasegawa PM amp Zhu JK (2002) Repressionof stress-responsive genes by FIERY2 a novel transcriptionalregulator in Arabidopsis Proceedings of the National Academyof Science of the USA 99 10899ndash10904

Received 29 November 2005 received in revised form 6 February2006 accepted for publication 6 February 2006

SUPPLEMENTARY MATERIAL

The following supplementary material is available for thispaper online

Table S1 (a) Genes up-regulated by ectopic AtCBF1expression in Poplar leaves The assigned gene identity(AGI) is based on homology to the Arabidopsis genomesequence (b) Genes up-regulated by ectopic AtCBF1expression in Poplar stems The assigned gene identity(AGI) is based on homology to the Arabidopsis genomesequence (c) Genes down-regulated by ectopic AtCBF1expression in Poplar leaves and stems The assigned geneidentity (AGI) is based on homology to the Arabidopsisgenome sequence

Table S2 Genes up-regulated by 7 d cold treatment inPoplar (leaf) The assigned function is based on homologyto the Arabidopsis genome sequence

Table S3 Genes up-regulated by 7 d cold treatment inPoplar (stem) The assigned function is based on homologyto the Arabidopsis genome sequence

Table S4 Normalized expression levels for orthologues tothe Arabidopsis CBF2 and CBF123 regulons The assignedgene identity (AGI) is based on homology to the Arabidop-sis genome sequence

Table S5 Positional analysis of DRE and ABRE distancesfrom ATG in Populus balsamifera subsp trichocarpa Theassigned gene identity (AGI) is based on homology to theArabidopsis genome sequence

Figure S1 Sequence alignments used to produce phyloge-netic tree in Fig 5

This material is available as part of the online article fromhttpwwwblackwell-synergycom

1260

C Benedict

et al



29

1259ndash1272

functions remain largely unknown (Svensson

et al

2002)Cold acclimation is better understood in herbaceous annu-als such as

Arabidopsis thaliana

where members of theC-repeat binding factor (CBF or DREB1) family of tran-scriptional activators which bind the

cis

-element known asthe C-repeat (CRT)dehydration-responsive element(DRE) (Stockinger Gilmour amp Thomashow 1997) havebeen shown to control the transcription of a suite of genesthat play important roles in cold acclimation and the devel-opment of FT The

Arabidopsis

CBFDREB1 family con-sists of six paralogues but only three [

CBF1

(

DREB1b

)

CBF2

(

DREB1c

) and

CBF3

(

DREB1a

)] are cold-induc-ible (Sakuma

et al

2002) These three low-temperature-responsive

CBF

s colocalize to an 87 kb region of chromo-some 4 and share a conserved AP2 DNA binding regionflanked by the characteristic amino acid motifsPKKPAGRxKFxETRHP and DSAWR (Jaglo

et al

2001)Based on a number of experiments using transgenic plantsat least 12 of the cold-induced transcriptional changesobserved in

Arabidopsis

are accounted for by the membersof the

CBF

transcription factor family (

CBF1-3

) (Fowleramp Thomashow 2002 Vogel

et al

2005 van Buskirk amp Tho-mashow 2006) and ectopic expression of

Arabidopsis

CBF1-4 has been shown to improve FT of non-acclimatedplants (Jaglo-Ottosen

et al

1998 Gilmour

et al

2000Haake

et al

2002 Gilmour Fowler amp Thomashow 2004)The accumulation of CBF transcripts and the activity of

the CRT regulatory motif in

Arabidopsis

is also modulatedby the presence and quality of light during cold stress (Kim

et al

2002 Fowler Cook amp Thomashow 2005) A shortperiod of exposure to red light has been shown to be suffi-cient to induce CRT-driven GUS reporter expression in thecold and this induction is photo-reversible with far-redlight GUS reporter expression is also altered in phyto-chrome mutant backgrounds suggesting that day lengthmediates expression of the CBF regulon through phyto-chrome B in

Arabidopsis

(Kim

et al

2002) Furthermorethe kinetics of endogenous CBF transcript accumulation isreduced in complete darkness (Kim

et al

2002) suggestingthat light regulation occurs at a point upstream of CBFexpression These data raise the question of whether CBFor related transcriptional regulators may be involved in theday length-mediated growth cessation in woody species Ithas been suggested that growth cessation in woody peren-nials mediated by short days and phytochrome operatesindependently of the low-temperature-driven acquisition ofdeep FT (Welling

et al

2002) However the observationthat pre-treatment with short days enhances the expressionof low temperature response genes (Puhakainen

et al

2004) and the finding that a CBF-like transcription factoris induced by short days in the cambial meristem of poplar(Schrader

et al

2004) indicates that there may be cross-talkbetween these two acclimation mechanisms

Following their original discovery in

Arabidopsis

the listof plants with cold-inducible

CBF

orthologues hasexpanded to include cold-sensitive and cold-tolerantannual species (Jaglo

et al

2001) and woody perennials(Kitashiba

et al

2002 Owens

et al

2002) Puhakainen

et al

(2004) have also demonstrated that AtCBF3 can activategene transcription via the birch

Bplti36

promoter sequencein transgenic

Arabidopsis

indicating that the structure andfunction of the active domain in the promoters of CBF-mediated cold-responsive genes have been conserved inherbaceous and woody perennials However detailed stud-ies of the role of CBF-related proteins in cold acclimationin tree species have been lacking Based on the demon-strated effects of both low temperature and light on CBFtranscript accumulation in

Arabidopsis

we have examinedhow CBF expression affected the FT of non-dormant pop-lar leaf and meristematic stem tissue under long-day growthconditions This experimental design enabled us to focus onthe role the CBF-mediated signalling pathway plays in thetemperature response of different tissues in a woody peren-nial without complicating interactions with day length anddormancy Our data demonstrate (1) that CBFs areinvolved in the FT mechanism of temperate trees speciessuch as poplar (2) that distinct regulons control stem (mer-istematic) versus leaf (non-meristematic) FT and (3) thatthe

Populus

genome contains multiple CBFs that are likelyto be differentially employed in perennial (stem) andannual (leaf) tissues

MATERIALS AND METHODS

Construction of transformation vector

A CaMV 35S

Promoter

Arabidopsis

CBF1 cDNA35S

Terminator

cassette (Jaglo-Ottosen

et al

1998) was ligated as a

Hind

IIIfragment into the binary vector pGAH (Onouchi

et al

1991) that had been linearised with

Hind

III

Poplar transformation

Plants of poplar clone 717-1B4 (

Populus tremula

times

alba

)were grown

in vitro

in a culture room at 24

deg

C under cool-white fluorescent lights (40ndash60

micro

mol m

minus

2

s

minus

1

16 h photope-riod) Stem internodes (7ndash10 mm in length) and leaf discs(5 mm in diameter) from fully expanded young leaves wereused as the explants for the transformation (DeBlock1990)

Agrobacterium tumefaciens

strain EHA105 carrying theplasmid pGAH35SAtCBF1 was grown overnight at 28

deg

Cin liquid YEP medium supplemented with 50 mg L

minus

1

hygro-mycin and 50 mg L

minus

1

kanamycin The cells were collectedby centrifugation at 1500 g for 20 min and resuspended toan OD

600

=

04ndash06 in Murashige and Skoog (MS) mediumwith 20

micro

g L

minus

1

acetosyringone (AS) Explants (stem intern-odes or leaf discs) were soaked for 10ndash20 min in the bacte-rial suspension incubated on a shaker (300 g) for 1 h atroom temperature followed by cultivation on callus induc-tion medium (CIM pH 52) supplemented with 20

micro

g L

minus

1

AS at 25

deg

C in the dark for 2ndash3 d After 2ndash3 d of cocultiva-tion explants were washed four times with double-distilledwater and once with wash solution (WS pH 58) supple-mented with 500 mg L

minus

1

cefotaxime The washed explantswere blotted dry with paper towels allowed to stand in a

The CBF regulon in

Populus

spp

1261



29

1259ndash1272


minus

1


minus

1


deg


















RESULTS






Leaves Stems

NA CA NA CA

TEL50











pGAH35SAtCBF1


LBRB

35SPNOSp HygR

NOST NOST

AtCBF118S

S L S L S L

Rel

ativ

em

RN

Aco

nten

tC

BF

118

S

S L

PtWT Line 1 Line 2

S LS L0

1

2

3

4L

L

AtWT

(a)

(b)

(c)






Plant age (weeks)

Pla

nthe

ight

(cm

)

0 1 2 3 4 5 60

5

10

15

20

25(d)

(a)

(b)

Line 1 WT

Line 1 WT

(c)













50

30

20

12

10

08

06

04

Rel

ativ

eex

pres

sion

(fol

d-ch

ange

)

AtCBF1 WT Cold

L SSL

A

B

C






CBF1Leaf FC

CBF1Stem FC

WTLeaf FC











(a)


AP2 domain

AtCBF2

AtCBF3

AtCBF1

BnCBF-C

BnCBF-A

BnCBF-B

AtCBF4

PtCBF1

PtCBF2

Nt111B

Nt111A

PtCBF3

PtCBF4

PaDREB1

AtDdf2

AtDdf1

PtCBFL1

PtCBFL2

PtDRTY

PtTINY-Like

AtTINY100

100

100

99

99

100

94

100

56

92

91 75

97

100 65

82

8663

(b)







DISCUSSION



Rel

ativ

eab

unda

nce


0 4 8 12 16 20 24

0

2

4

6StemLeaf

0

2

4

6PtCBF1(a) (b)

(c) (d)

0

2

4

6PtCBF3

PtCBF2

PtCBF4

Relative

abundance

0 4 8 12 16 20 2400

02

04

06

08

10

12

14













ACKNOWLEDGMENTS



REFERENCES

















































































The CBF regulon in

Populus

spp

1261



29

1259ndash1272


minus

1


minus

1


deg


















RESULTS






Leaves Stems

NA CA NA CA

TEL50











pGAH35SAtCBF1


LBRB

35SPNOSp HygR

NOST NOST

AtCBF118S

S L S L S L

Rel

ativ

em

RN

Aco

nten

tC

BF

118

S

S L

PtWT Line 1 Line 2

S LS L0

1

2

3

4L

L

AtWT

(a)

(b)

(c)






Plant age (weeks)

Pla

nthe

ight

(cm

)

0 1 2 3 4 5 60

5

10

15

20

25(d)

(a)

(b)

Line 1 WT

Line 1 WT

(c)













50

30

20

12

10

08

06

04

Rel

ativ

eex

pres

sion

(fol

d-ch

ange

)

AtCBF1 WT Cold

L SSL

A

B

C






CBF1Leaf FC

CBF1Stem FC

WTLeaf FC











(a)


AP2 domain

AtCBF2

AtCBF3

AtCBF1

BnCBF-C

BnCBF-A

BnCBF-B

AtCBF4

PtCBF1

PtCBF2

Nt111B

Nt111A

PtCBF3

PtCBF4

PaDREB1

AtDdf2

AtDdf1

PtCBFL1

PtCBFL2

PtDRTY

PtTINY-Like

AtTINY100

100

100

99

99

100

94

100

56

92

91 75

97

100 65

82

8663

(b)







DISCUSSION



Rel

ativ

eab

unda

nce


0 4 8 12 16 20 24

0

2

4

6StemLeaf

0

2

4

6PtCBF1(a) (b)

(c) (d)

0

2

4

6PtCBF3

PtCBF2

PtCBF4

Relative

abundance

0 4 8 12 16 20 2400

02

04

06

08

10

12

14













ACKNOWLEDGMENTS



REFERENCES























































































RESULTS






Leaves Stems

NA CA NA CA

TEL50











pGAH35SAtCBF1


LBRB

35SPNOSp HygR

NOST NOST

AtCBF118S

S L S L S L

Rel

ativ

em

RN

Aco

nten

tC

BF

118

S

S L

PtWT Line 1 Line 2

S LS L0

1

2

3

4L

L

AtWT

(a)

(b)

(c)






Plant age (weeks)

Pla

nthe

ight

(cm

)

0 1 2 3 4 5 60

5

10

15

20

25(d)

(a)

(b)

Line 1 WT

Line 1 WT

(c)













50

30

20

12

10

08

06

04

Rel

ativ

eex

pres

sion

(fol

d-ch

ange

)

AtCBF1 WT Cold

L SSL

A

B

C






CBF1Leaf FC

CBF1Stem FC

WTLeaf FC











(a)


AP2 domain

AtCBF2

AtCBF3

AtCBF1

BnCBF-C

BnCBF-A

BnCBF-B

AtCBF4

PtCBF1

PtCBF2

Nt111B

Nt111A

PtCBF3

PtCBF4

PaDREB1

AtDdf2

AtDdf1

PtCBFL1

PtCBFL2

PtDRTY

PtTINY-Like

AtTINY100

100

100

99

99

100

94

100

56

92

91 75

97

100 65

82

8663

(b)







DISCUSSION



Rel

ativ

eab

unda

nce


0 4 8 12 16 20 24

0

2

4

6StemLeaf

0

2

4

6PtCBF1(a) (b)

(c) (d)

0

2

4

6PtCBF3

PtCBF2

PtCBF4

Relative

abundance

0 4 8 12 16 20 2400

02

04

06

08

10

12

14













ACKNOWLEDGMENTS



REFERENCES

























































































pGAH35SAtCBF1


LBRB

35SPNOSp HygR

NOST NOST

AtCBF118S

S L S L S L

Rel

ativ

em

RN

Aco

nten

tC

BF

118

S

S L

PtWT Line 1 Line 2

S LS L0

1

2

3

4L

L

AtWT

(a)

(b)

(c)






Plant age (weeks)

Pla

nthe

ight

(cm

)

0 1 2 3 4 5 60

5

10

15

20

25(d)

(a)

(b)

Line 1 WT

Line 1 WT

(c)













50

30

20

12

10

08

06

04

Rel

ativ

eex

pres

sion

(fol

d-ch

ange

)

AtCBF1 WT Cold

L SSL

A

B

C






CBF1Leaf FC

CBF1Stem FC

WTLeaf FC











(a)


AP2 domain

AtCBF2

AtCBF3

AtCBF1

BnCBF-C

BnCBF-A

BnCBF-B

AtCBF4

PtCBF1

PtCBF2

Nt111B

Nt111A

PtCBF3

PtCBF4

PaDREB1

AtDdf2

AtDdf1

PtCBFL1

PtCBFL2

PtDRTY

PtTINY-Like

AtTINY100

100

100

99

99

100

94

100

56

92

91 75

97

100 65

82

8663

(b)







DISCUSSION



Rel

ativ

eab

unda

nce


0 4 8 12 16 20 24

0

2

4

6StemLeaf

0

2

4

6PtCBF1(a) (b)

(c) (d)

0

2

4

6PtCBF3

PtCBF2

PtCBF4

Relative

abundance

0 4 8 12 16 20 2400

02

04

06

08

10

12

14













ACKNOWLEDGMENTS



REFERENCES






















































































Plant age (weeks)

Pla

nthe

ight

(cm

)

0 1 2 3 4 5 60

5

10

15

20

25(d)

(a)

(b)

Line 1 WT

Line 1 WT

(c)













50

30

20

12

10

08

06

04

Rel

ativ

eex

pres

sion

(fol

d-ch

ange

)

AtCBF1 WT Cold

L SSL

A

B

C






CBF1Leaf FC

CBF1Stem FC

WTLeaf FC











(a)


AP2 domain

AtCBF2

AtCBF3

AtCBF1

BnCBF-C

BnCBF-A

BnCBF-B

AtCBF4

PtCBF1

PtCBF2

Nt111B

Nt111A

PtCBF3

PtCBF4

PaDREB1

AtDdf2

AtDdf1

PtCBFL1

PtCBFL2

PtDRTY

PtTINY-Like

AtTINY100

100

100

99

99

100

94

100

56

92

91 75

97

100 65

82

8663

(b)







DISCUSSION



Rel

ativ

eab

unda

nce


0 4 8 12 16 20 24

0

2

4

6StemLeaf

0

2

4

6PtCBF1(a) (b)

(c) (d)

0

2

4

6PtCBF3

PtCBF2

PtCBF4

Relative

abundance

0 4 8 12 16 20 2400

02

04

06

08

10

12

14













ACKNOWLEDGMENTS



REFERENCES

























































































50

30

20

12

10

08

06

04

Rel

ativ

eex

pres

sion

(fol

d-ch

ange

)

AtCBF1 WT Cold

L SSL

A

B

C






CBF1Leaf FC

CBF1Stem FC

WTLeaf FC











(a)


AP2 domain

AtCBF2

AtCBF3

AtCBF1

BnCBF-C

BnCBF-A

BnCBF-B

AtCBF4

PtCBF1

PtCBF2

Nt111B

Nt111A

PtCBF3

PtCBF4

PaDREB1

AtDdf2

AtDdf1

PtCBFL1

PtCBFL2

PtDRTY

PtTINY-Like

AtTINY100

100

100

99

99

100

94

100

56

92

91 75

97

100 65

82

8663

(b)







DISCUSSION



Rel

ativ

eab

unda

nce


0 4 8 12 16 20 24

0

2

4

6StemLeaf

0

2

4

6PtCBF1(a) (b)

(c) (d)

0

2

4

6PtCBF3

PtCBF2

PtCBF4

Relative

abundance

0 4 8 12 16 20 2400

02

04

06

08

10

12

14













ACKNOWLEDGMENTS



REFERENCES






















































































CBF1Leaf FC

CBF1Stem FC

WTLeaf FC











(a)


AP2 domain

AtCBF2

AtCBF3

AtCBF1

BnCBF-C

BnCBF-A

BnCBF-B

AtCBF4

PtCBF1

PtCBF2

Nt111B

Nt111A

PtCBF3

PtCBF4

PaDREB1

AtDdf2

AtDdf1

PtCBFL1

PtCBFL2

PtDRTY

PtTINY-Like

AtTINY100

100

100

99

99

100

94

100

56

92

91 75

97

100 65

82

8663

(b)







DISCUSSION



Rel

ativ

eab

unda

nce


0 4 8 12 16 20 24

0

2

4

6StemLeaf

0

2

4

6PtCBF1(a) (b)

(c) (d)

0

2

4

6PtCBF3

PtCBF2

PtCBF4

Relative

abundance

0 4 8 12 16 20 2400

02

04

06

08

10

12

14













ACKNOWLEDGMENTS



REFERENCES




















































































(a)


AP2 domain

AtCBF2

AtCBF3

AtCBF1

BnCBF-C

BnCBF-A

BnCBF-B

AtCBF4

PtCBF1

PtCBF2

Nt111B

Nt111A

PtCBF3

PtCBF4

PaDREB1

AtDdf2

AtDdf1

PtCBFL1

PtCBFL2

PtDRTY

PtTINY-Like

AtTINY100

100

100

99

99

100

94

100

56

92

91 75

97

100 65

82

8663

(b)







DISCUSSION



Rel

ativ

eab

unda

nce


0 4 8 12 16 20 24

0

2

4

6StemLeaf

0

2

4

6PtCBF1(a) (b)

(c) (d)

0

2

4

6PtCBF3

PtCBF2

PtCBF4

Relative

abundance

0 4 8 12 16 20 2400

02

04

06

08

10

12

14













ACKNOWLEDGMENTS



REFERENCES























































































DISCUSSION



Rel

ativ

eab

unda

nce


0 4 8 12 16 20 24

0

2

4

6StemLeaf

0

2

4

6PtCBF1(a) (b)

(c) (d)

0

2

4

6PtCBF3

PtCBF2

PtCBF4

Relative

abundance

0 4 8 12 16 20 2400

02

04

06

08

10

12

14













ACKNOWLEDGMENTS



REFERENCES





























































































ACKNOWLEDGMENTS



REFERENCES





















































































ACKNOWLEDGMENTS



REFERENCES














































































































































































Documents

The CBF1-dependent low temperature signalling … BHALERAO 3, NORMAN P. A. HUNER 4, CHAD E. FINN 2,5, TONY H. H. CHEN 2 & VAUGHAN HURRY 1 1Umeå Plant Science Centre, Department of