Original Paper

Abstract: The effects of cold acclimation on primary metab-olism in actively growing poplar (Populus tremula L. × P. tremu-loides Michaux) were studied. Three-month-old poplar plantswere exposed to chilling stress (4 �C) and compared to plant ma-terial kept at a control temperature (23 �C). This treatment didnot affect the survival of the plants but growth was almoststopped. The freezing tolerance of the adult leaves increasedfrom – 5.7 �C for the control plants to – 9.8 �C after 14 days ofexposure to 4 �C. During acclimation, the evolution of solublecarbohydrate contents was followed in the leaves. Sucrose, glu-cose, fructose and trehalose accumulated rapidly under chillingconditions, while raffinose content increased after one weekat 4 �C. Proteomic analyses, by bidimensional electrophoresis,performed during this stage revealed that a large number ofproteins had higher expression, while much less proteins dis-appeared or had a lower abundance. MALDI-TOF-MS analysesenabled ca. 30 spots to be proposed for candidate proteins.Among the accumulating or appearing proteins proposed,about a third presented similarities with chaperone-like proteins(heat shock proteins, chaperonins). In addition, dehydrins andother late embryogenesis abundant proteins, i.e., stress-respon-sive proteins, detoxifying enzymes, proteins involved in stresssignalling and transduction pathways were also activated ornewly synthesised. Finally, cold exposure induced a decrease inthe candidate proteins involved in cell wall or energy produc-tion.

Key words: Populus tremula × P. tremuloides, cold acclimation,freezing tolerance, trehalose, raffinose, proteomics.

Abbreviations:2DE: bidimensional electrophoresisFW: fresh weightHSP: heat shock proteinsIPG: immobilized pH gradientLEA: late embryogenesis abundant proteinsLT50: temperature inducing 50% of injured cellsRFOs: raffinose family oligosaccharides

Introduction

Temperature is a major environmental constraint influencingthe distribution, growth, development and productivity ofcrops, horticultural plants and wild species. A common featureof many plant species exposed to low non-freezing tempera-tures, from 0 to 15 �C, is the phenomenon of chilling stress.For plants adapted to temperate climates, exposure to lowtemperatures induces several physiological changes that allowthem to withstand this stress (Levitt, 1980). The positive ef-fects of these changes induced by chilling stress are referredto as cold acclimation. This results in a lowering of the tem-perature at which the plant is damaged or killed by chillingor freezing temperatures (Graham and Patterson, 1982; Guy,1990; Levitt, 1980). It involves a reduction or cessation ofgrowth, the production of reactive oxygen species (Prasad etal., 1994) and a reduction of water tissue content (Imanishi etal., 1998). Additionally, cold acclimation is associated with amodification of gene expression, mainly regulated at the ini-tiation of transcription (e.g., activation or repression of specificcold-responsive genes, CBF/DREB1…) (Chen et al., 2002; Guy,1990; Pearce, 1999; Seki et al., 2001), the appearance or ac-cumulation of proteins such as antifreeze proteins (Griffith etal., 1997), heat shock proteins (HSP) (Wisniewski et al., 1996),stable protein 1 (Wang et al., 2002) or late embryogenesisabundant proteins (LEA) (Antikainen and Griffith, 1997), theaccumulation of different compounds such as carbohydrates,amino acids, sugar alcohols or pigments (Krause et al., 1999)and other stress-related molecules such as polyamines (Bou-chereau et al., 1999) or antioxidants (Hausman et al., 2000;Iba, 2002).

Proteomics is a recent addition to the molecular tools used toanalyse stress responses in plants (Salekdeh et al., 2002). Itprovides a direct assessment of proteins involved in stress re-sponse pathways and consequently is a valuable link betweenthe classical physiological approach and molecular tools. Theforthcoming publication of the poplar genome will drasticallyincrease the power of proteomics in studies involving woodyspecies by allowing more precise identification of polypep-tides obtained by MALDI-TOF-MS.

Until recently, studies on cold acclimation have been mainlyundertaken in herbaceous species. Moreover, the effects ofcold have been much less studied in woody plants, althoughthese species have to survive for decades. Most temperate

Responses of Poplar to Chilling Temperatures:Proteomic and Physiological Aspects

J. Renaut1,2, S. Lutts2, L. Hoffmann1, and J.-F. Hausman1

1 CRP-Gabriel Lippmann, CREBS, Luxembourg, GD Luxembourg2 Université Catholique de Louvain-la-Neuve, Unité de Biologie Végétale, 5 (Bte 13) Place Croix du Sud,

1348 Louvain-la-Neuve, Belgium

Received: September 10, 2003; Accepted: December 12, 2003

Plant Biology 6 (2004): 81–90© Georg Thieme Verlag Stuttgart · New YorkISSN 1435-8603 · DOI 10.1055/s-2004-815733

81

woody plants have an annual cycle alternating between activegrowth and winter rest or dormancy (Arora et al., 1997). How-ever, trees can also face chilling stress, especially during earlyspring growth or when autumnal bud set is delayed. In thiscontext, the dynamic comparison of physiological and proteo-mic changes between acclimated and non-acclimated leaves,i.e., frost-sensitive organs, is of importance to understand thestress coping mechanisms in trees.

Poplar trees are fast-growing species. They constitute a majorsegment of the plant population in temperate forests in thenorthern hemisphere and are important both economicallyfor timber or pulp production, and as a major component ofthe ecosystem (Bradshaw and Stettler, 1995; Pelah et al.,1997). Poplars are easy to propagate by root or stem cuttings,and the availability of clonal material facilitates the testing ofidentical genotypes under different conditions (Howe et al.,1999).

The present study aims to depict the interacting processes in-volved in cold acclimation in actively growing poplars throughmorphological, physiological and proteomic approaches. It at-tempts to evaluate and compare the cold acclimation responseof cuttings of Populus tremula × P. tremuloides, studying freez-ing tolerance, growth and carbohydrate content evolution inacclimated and non-acclimated plants. Proteomics also offersnew possibilities as regards stress tolerance strategies andemerges as the technology of the post-genomic era. The com-bination of proteomic and physiological approaches allowsprecise correlations to be made between biochemical or phys-iological observations and changes in proteomic patterns andthus constitutes a link between molecular biology and phys-iology.

Materials and Methods

Plant material

A clone of Populus tremula L. × P. tremuloides Michaux cv.Muhs 1 was used in the experiments. The plant material wascultivated in vitro in polypropylene jars on a Murashige andSkoog (1962) medium supplemented with 0.3mg l–1 benzyl-aminopurine as a growth regulator. After rooting, the plantswere acclimated in a mixture of peat moss and sand (5 :1)and grown in a greenhouse for 3 months at approximately23 �C/18 �C (day/night) and a minimum photoperiod of 16 h.

Whole plants (8 per condition and per experiment) measuringca. 80 cmwere pre-acclimated in growth chambers with a 16 hphotoperiod and a light intensity of 150 µmol m–2 s–1 (SylvaniaGrolux Fluorescent lamps) for one week. Afterwards, chilling(4 �C) or control (23 �C) temperatures were applied for 14 days.Fully expanded leaves (from 7 to 11) were sampled for carbo-hydrate analyses on days 0, 1, 2, 3, 4, 7, 10 and 14, around11:00 a.m. One hundred µg fresh weight of these leaves werefrozen in liquid nitrogen and preserved at – 80 �C until furtheranalyses were carried out. Similar sampling was undertakenfor protein analyses on day 7 and 14 for both stress and controlconditions.

Freezing tolerance

Freezing tolerancewas assessed in order to evaluate the abilityof the tissue studied (i.e., fully expanded poplar leaves) to ac-climate to cold. Freezing tolerance was determined after atreatment period of 0, 4, 7 and 14 days at 4 �C by measuringchanges in electrical conductivity according to Arora et al.(1996), with slight modifications. Entire leaves were cut fromcontrol and stressed plants and put in empty tubes. Eightleaves were used for each freezing temperature assay (0, – 3,– 6, – 9, – 12, – 15 and – 18 �C). Timing conditions for the cool-ing rate and shaking, as well as sampling conditions and con-ductimeter calibration were determined in order to minimizevariations during measurements. Samples were cooled at arate of 5 �C h–1 in a temperature-controlled bath (Julabo Labor-technik, F12-MP) initially maintained at 5 �C. Tubes were re-moved at a selected temperature and allowed to reach roomtemperature; then 40ml of double distilled water were added.Tubeswere shaken for 2 h at room temperature and conductiv-ity was measured with Cynerscan CON400 (Eutech Instru-ment™). Tubes containing leaves and water were autoclaved(0.12 MPa, 120 �C, 20min) and, after cooling, conductivity wasagainmeasured. The percentage of ion leakage and injuryweredetermined according to Arora et al. (1996). From plotting thepercentage of injury as a function of treatment temperature,the temperature at which 50% of the cells were injured wasobtained; this temperature was defined as LT50. A sigmoid re-gression (3 parameters) was applied to the set of points. Thevalidation of the results was performed with an ANOVA, fol-lowed by a t-test.

Survival and growth

Plant survival was estimated after 14 days of treatment in bothcontrol and cold conditions. Survival was determined as thepercentage of plants not affected by shoot necrosis. Measuringelongation of the plant stems during 14 days of culture wasused to assess the growth of poplar plants.

Determination of carbohydrate content

Samples of leaves (approximately 100mg FW) were ground inliquid nitrogen and extracted with 1ml 80% ethanol for 1 hwith gentle shaking. After centrifugation at 10000 × g at 4 �Cfor 10min, the supernatant was dried by vacuum centrifuga-tion at 40 �C. Samples were re-suspended in reverse osmosiswater prior to analysis using high-performance anion ex-change chromatography coupled with pulsed amperometricdetection. The analyses were performed on a Dionex chroma-tography system. Mono- and disaccharides were separated ona Carbo-Pac PA10 (Dionex) guard column (4 × 50mm) and a se-rial Carbo-Pac PA10 (Dionex) column (4 × 250mm) at a flowrate of 1ml min–1 using 18mM NaOH as eluent. Detectionwas made by triple pulsed amperometry with a gold workingelectrode, according to Wilson et al. (1995). External standardcalibration was performed using a calibration solution at thebeginning of every batch.

Protein extraction and 2DE

The extraction procedure was based on that of Görg et al.(2000) with some modifications. Leaf tissue samples (5 rep-licates) were ground in liquid nitrogen and suspended in

Plant Biology 6 (2004) J. Renaut et al.82

20% w/v TCA in acetone with 0.07% w/v 2-mercaptoethanol at– 20 �C for 1 h, followed by centrifugation for 15min at35000 × g and 4 �C. The pellets werewashed with ice-cold ace-tone containing 0.07% 2-mercaptoethanol, incubated at – 20 �Cfor 1 h and centrifuged again at 4 �C. This step was repeatedthree times and the pellets were freeze-dried. The samplepowders were then solubilised in lysis buffer (8M urea, 2%w/v CHAPS, 2% w/v ampholytes IPG Buffer pH 4–5, pH 4.5–5.5 or 5–6 (Amersham Biosciences), 1mM PMSF, 1% w/v di-thiothreitol) and the protein concentration was determinedusing a quantification kit (2D Quant Kit, Amersham Biosci-ences) and BSA as standard.

Two-dimensional analysis (2DE) was carried out as describedby Görg et al. (2000, 2001) with minor modifications. For ana-lytical gels, the 24 cm immobilized pH gradient (IPG) stripswith a short pH range (pH 4–5, 4.5–5.5 and 5–6) were cho-sen, since identification of proteins obtained from non-se-quenced genomes (by MALDI-TOF MS) requires enhanced sep-aration of mixed proteins, both to avoid contamination by oth-er spots and to rely on a precise value of proteins pI. The gelswere rehydrated overnight with 450 µl of rehydration buffer(8M urea, 0.5% CHAPS, 0.2% w/v di-thiothreitol, 0.5% v/v IPGbuffers, 0.002% w/v bromophenol blue) in a reswelling tray(Amersham Biosciences). One hundred and seventy µg of pro-teins were loaded on each strip. Isoelectrofocusing was con-ducted at 20 �C with an IPG-Phor system (Amersham Biosci-ences). The running conditions were successively 200 V for80min, 500 V (80min), 1000V (80min) and finally 8000 V for11 h. The focused strips were equilibrated twice for 15min in10ml equilibration solution. The first equilibration was per-formed in a solution containing 50mM Tris-HCl, pH 8.8, 6Murea, 30% v/v glycerol, 2% w/v SDS, 0.002% w/v bromophenolblue and 1% w/v di-thiothreitol. The second equilibrationwas performed in a solution modified by the replacement ofdi-thiothreitol by 2.5% w/v iodoacetamide. Separation in thesecond dimension was performed in a vertical slab of acryl-amide (12.5% total monomer, with 2.6% crosslinker, gel size:25.5 × 19.6 × 0.1 cm, Amersham Biosciences) using an Ettan-DALTsix system (Amersham Biosciences). The protein spotswere visualised by staining with silver nitrate, without usingglutaraldehyde, essentially as described by Blum et al. (1987).

Image and data analysis

Silver stained gels were scanned using a calibrated Image-scanner II (Amersham Biosciences) at a resolution of 300 dpi(16 bits). Image treatment, spot detection and protein quan-tification were carried out using the ImageMaster 2D Elite(v.2003.02, Amersham Biosciences). The molecular masses ofprotein on gels were determined by the co-electrophoresis ofstandard protein markers (Amersham Biosciences) and pI ofthe proteins were determined by migration of the protein

spots on 24 cm IPG (pH 4–5, 4.5–5.5 and 5–6) strips. One2DE gel per plant was run and the percentage volume of eachspot was estimated and analysed. To avoid false positives, se-lected spots have to be present on 5 2DE gels to be validated(coming from5 plants from the same treatment). All spots pre-senting more than a 3-fold increase or a 3-fold decrease intheir volume (expressed in integrated optical density), or spotsthat were present or absent in one of the conditions were se-lected for mass spectrometry analyses.

Protein identification and database search

Selected protein spots were excised from 2DE gels and de-stained according to Gharahdaghi (1999). The gel pieces werethen digested with trypsin and analysed by MALDI-TOF-MSusing a Voyager-DE Elite Biospectrometry workstation (Per-Septive Biosystems, Applied Biosystems). For protein identifi-cation by peptide mass, the approach used was to searchagainst the SWISS-PROT and TrEMBL databases using PeptI-dent software (www.expasy.ch/tools/peptident.html) withViridiplantae as taxonomic category. Database queries werecarried out for monoisotopic peptide masses using the follow-ing parameters: peptide mass tolerance of ± 50 ppm; maxi-mum number of missed tryptic cleavages: 1. Accepted modifi-cations included the oxidation of Met, the conversion of N-ter-minal Gln to pyro-Gln and the acetylation of the N-terminus.Cys residues were assumed to have been reduced and alkylat-ed by iodoacetamide to carboxyamidomethyl cysteine.

Statistical analysis

Five independent experiments were carried out. A statisticalanalysis of daily growth rate results was carried out with at-test on SigmaStat software 2.0. Other results were statisti-cally analysed with a one-way variance analysis (ANOVA) orAnova on ranks when the normality test or equal variance testfailed. A Tukey multiple comparison test was performedwhensignificant differences were detected between treatments.Pearson’s correlation coefficient and ANOVA were carried outon Instat software 2.01 for MacIntosh.

Table 1 Daily growth rate and survival after 14 days of treatment at23�C and 4 �C of poplar plants (means ± SD, n = 12). A significant differ-ence was observed between treatments (p < 0.001)

Temperature Daily growth rate Survival

23�C 7.0 ± 1.3mm 100%4 �C 0.3 ± 0.3mm 100%

0 4 7

Days of treatment

10 14–12

–10

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a

b

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r2 = 0.993

Fig.1 Change in LT50 during the 14 days of treatment (4 �C vs. 23 �C).Letters indicate statistical differences between values (p < 0.001). r2

represents the coefficient of linearity.

Chilling Effects on Poplar Plant Biology 6 (2004) 83

Results

Survival and growth

Chilling at 4 �C did not affect the survival of poplar plants, sinceleaf necrosis was not observed (Table 1). Low temperature,however, had a negative effect on the growth of poplar plants:during the first 14 days of treatment, the daily growth rate ofplants kept at 4 �C (0.3mm per day; Table 1) was significantlylower (p < 0.001) than that of control plants (7.0mm per day)during the same period of time.

Freezing tolerance

The freezing tolerance of poplar was measured for non-accli-mated plants (grown at 23 �C) and for those that were exposedto cold treatment (Fig.1). The LT50 of non-acclimated plantswas – 5.75 �C and decreased linearly as a function of the dura-tion of treatment at 4 �C (r2 = 0.993). Indeed, plants that werecold-treated for 4, 7, 10 and 14 days showed a sharp decrease(t-test, p < 0.001) in their LT50 value to – 7.10 �C, – 8.00 �C,– 8.65 �C and – 9.80 �C, respectively.

Soluble carbohydrate content

Five different carbohydrates were analysed, namely sucrose,fructose, glucose, raffinose and trehalose. Their contentsmeas-ured at 23 �C did not showany significant changes. During coldtreatment, soluble carbohydrate contents varied to differentextents depending on the nature of the carbohydrate consid-ered. The most abundant soluble carbohydrates were glucose,fructose and sucrose (Fig. 2). They accumulated significantly incold-stressed plants. The level of sucrose (Fig. 2a) increasedsignificantly after 2 days of chilling, in comparison with con-trol leaves, and then remained stable at a significantly higherlevel until day 14. Fructose and glucose contents (Figs. 2b,c,respectively) showed a similar trend: a significant accumula-tion followed by a higher, although fluctuating, content untilday 14. At 4 �C, the raffinose content did not vary during thefirst 4 days (Fig. 3a) then started to accumulate linearly. After14 days of cold exposure, its level was almost four times thecontrol level. The raffinose level presented a very high corre-lation with LT50 (Pearson’s correlation, r = – 0.97, p = 0.05). The

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Fig. 2 Evolution of carbohydrate contents during chilling treatmentin poplar leaves: (a) sucrose; (b) fructose; and (c) glucose (n = 20). Let-ters indicate statistical differences between 4 �C values (from day 0until day 14).

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Fig. 3 Evolution of carbohydrate contents during chilling treatmentin poplar leaves: (a) raffinose; (b) trehalose (n = 20). Letters indicatestatistical differences between 4 �C values (from day 0 until day 14).

Plant Biology 6 (2004) J. Renaut et al.84

Fig. 4 2DE gel analyses of proteins extracted from poplar leaves sub-mitted to cold after 14 days (a–c) and control plants kept at 23�C(d–f). In the first dimension (isoelectrofocusing), 170 µg of proteinwere loaded on a 24 cm IPG strip with a linear gradient of pH 4–5

(a,d), pH 4.5–5.5 (b,e) or pH 5–6 (c, f). In the second dimension,12.5% SDS-PAGE gels were used. Proteins were visualised by silverstaining. The arrows indicate 60 proteins that changed reproduciblyand significantly in cold-exposed poplars compared with controls.

Chilling Effects on Poplar Plant Biology 6 (2004) 85

trehalose content (Fig. 3b) of cold-treated leaves increasedafter day 2 then reached a plateau.

2DE and protein identification by MS

After 2DE gel separation and silver staining, more than 1500poplar leaf proteins were detected by digital image analysis;at least 800 gave reproducible staining patterns for all sam-ples, as judged by spot intensity ranking using ImageMaster2D Elite software. Sixty protein spots showed significant andreproducible changes in abundance in at least one stressingcondition analysed (i.e., in the case of protein analyses, 7 or14 days of cold exposure) compared with controls (plants keptat 23 �C). Fig. 4 shows the position of the 60 proteins on 2DEgels from poplar leaves. Among these 60 proteins, 26 could be

identified. The others were either predicted proteins with un-known functions or their identificationwas not possible due tounreliable results (not enough peptides matched or MW and/or pI out of range). Peptide mass fingerprinting with MALDI-TOF-MS identified 26 proteins with homologies with pro-teins of other organisms found in databases (Table 2). Proteinsappearing, or with an increased expression, at 4 �C can beclassified according to their function and/or their role in stressresponses. The first group includes chaperone-like proteins(Table 2: spots 3, 4, 6, 7, 8, 24, 39, 40, 55 and 62). Two of theseproteins were found only in cold-treated samples (Fig. 5: spots24 and 62) while others accumulated, sometimes transiently(Fig. 5a: spots 6, 7 and 55). The second cluster represents theproteins usually described as drought-responsive (Table 2,Fig. 5b: spots 21 and 60). A third class comprises proteins

Table 2 Proteins identified by peptide mass fingerprinting

Spot identification on2DE gels

Spot identification by MALDI-TOF-MS analysis and database search

Spot pI MW Identity (organism) Matcha Sequencecoverageb

pI MW Accessionnumberc

3 5.11 69900 Heat shock protein 70 (Lilium longiflorum) 5 10.2% 5.17 70997 Q406934 5.15 69450 Heat shock protein 70 (Daucus carota) 5 8.1% 5.15 72051 P267916 5.32 62100 Heat shock protein 70 (Dunaliella salina) 4 9.2% 5.31 71008 Q8RY447 5.13 60300 Chaperonin CPN60-1 (Zea mays) 5 22.1% 5.24 61211 P291858 5.37 63900 Cytosolic heat shock 70 protein (Spinacea oleracea) 5 8.3% 5.09 71493 O22664

16 5.25 41000 F20D23.25-prenyl transferase-like protein(Arabidopsis thaliana)

4 12.7% 5.21 41916 Q9SHG4

17 5.32 34150 Annexin (Nicotiana tabacum) 3 18.2% 5.38 35949 O2413218 5.92 34000 Alcohol dehydrogenase (Arabidopsis thaliana) 5 16.6% 6.51 40698 Q9653319 5.74 27750 Ascorbate peroxidase (Pimpinella brachycarpa) 3 14.4% 5.69 27747 Q9SED020 5.73 28700 Putative RAB24 Protein [Peroxiredoxin-like] (Oryza sativa) 6 32.7% 5.42 24232 Q8GVG921 5.34 25400 Dehydrin (Phaseolus vulgaris) 3 18.3% 5.32 22973 Q4111123 5.48 23400 CRT/DRE binding factor 1 (Hordeum vulgare) 3 11.5% 5.27 23137 Q8S4R324 5.13 22500 Small Heat shock protein [HSP20 class III – chloroplastic]

(Pisum sativum)4 45.2% 5.15 21287 P09886

34 4.83 85000 MAPK3-like protein kinase (Arabidopsis thaliana) 4 3.4% 4.79 87426 O2319037 4.29 54000 Putative thioredoxin (Arabidopsis thaliana) 5 15.5% 5.01 53115 Q9ZPH239 4.80 68400 Heat shock cognate 70 kDa protein 1

(Arabidopsis thaliana)3 10.3% 5.03 71357 P22953

40 4.75 68900 Heat shock protein 70 [precursor] (Citrullus vulgaris) 7 16.1% 5.05 68383 O0405641 4.91 57700 UDP-glucose dehydrogenase, putative

(Arabidopsis thaliana)9 20.2% 5.58 53174 Q8L8N1

42 4.62 56800 Rubisco large subunit [precursor] (Populus tremuloides) 8 23.5% – 53160 P4871346 4.97 49000 UTP-glucose-1-phosphate uridylyltransferase

(Hordeum vulgare)8 18% 5.2 51783 Q43772

47 5.14 49000 ATP synthase beta unit (Tapiscia sinensis) 11 35% 5.13 50994 Q95FJ248 5.22 46800 Glucosyltransferase (Arabidopsis thaliana) 8 21% 5.26 51265 O2338055 4.96 22000 Heat shock protein 22 kDa, mitochondrial

(Pisum sativum)6 28.2% 4.95 20332 P09886

59 5.07 8500 MAPK-like protein [fragment] (Oryza sativa) 4 14.7% – 7500 Q9MB0160 4.74 10200 Late Embryogenesis Abundant protein 10 kDa

(Helianthus annuus)4 48.9% 5.36 10036 P46514

62 4.99 27700 Heat shock related protein 70 [fragment](Helianthus annuus)

3 15.2% 4.52 29079 O04223

a Number of peptides matched. b Sequence percentage coverage. c Accessionnumber in TrEMBL or SWISS-PROT databases.Proteins from leaves were extracted and separated by 2DE within a pH range of4 to 6. The protein spots were excised from the gels, digested and the peptides

generated and analysed by MALDI-TOF-MS. Search in SWISS-PROT and TrEMBLdatabases resulted in the proposed candidate proteins listed in the table. Forprotein search, a window of mass tolerance of 50 ppm was used.

Plant Biology 6 (2004) J. Renaut et al.86

involved in stress signalling and transduction (Table 2, Fig. 5c:spots 17, 23, 34 and 59). Enzymes involved in detoxifying path-ways constitute another cluster of proteins (Table 2, Fig. 5d:spots 19, 20 and 37). Finally two proteins could not be associ-ated with stress response mechanisms (Table 2, Fig. 5e: spots16 and 18). Proteins disappearing or with a decreased ex-pression at 4 �C were mostly involved in energy metabolism(Table 2, Fig. 6: spots 41, 42, 46, 47 and 48).

Discussion

Poplar has a great ability to withstand different environmentalstresses. In this study, 80-cm tall plants of Populus tremula × P.tremuloides were submitted to a chilling temperature treat-ment during their active growth. Fully expanded poplar leaveswere monitored in an attempt to characterise the changes incarbohydrate metabolism during the cold acclimation process.The cuttings or, more precisely, their leaves, were able to accli-mate to cold, as shown by increased freezing tolerance whenplants were exposed to a treatment at 4 �C (Fig.1). Similar re-sults were obtained in cell suspension cultures of peach (AroraandWisniewski, 1995), in apices of birch (Rinne et al., 1999) orin cuttings of poplar (Hausman et al., 2000). This cold acclima-tion indicates that poplar is able to develop efficient tolerancemechanisms and that the temperature constraints imposed orthe leaves can be considered as a mild stress (Lichtenthaler,1996).

Contrary to survival, which was not affected by the chillingtreatment, the stem elongation of cold-stressed poplars wasalmost stopped (Table 1). Previous studies showed that thefresh weight of in vitro poplar plants cultured for 14 days at10 �C was significantly lower in comparison with those kept at23 �C (Hausman et al., 2000). The slowdown in growth may bedue tomobilisation of carbohydrates for a putative cryoprotec-tive process.

In fact, almost all the monitored carbohydrates observed accu-mulated in the leaves when plants were exposed to chillingstress (Figs. 2,3 ). Glucose, fructose and sucrose are essentialfor plant growth, maintenance and repair processes and plantsurvival, also being major sources of cellular energy. Hence,the increase in sucrose and its galactosides (Fig. 2) observedin the poplar leaves exposed to cold confirmed that this treat-ment induced cellular perturbations. These observations arenot new (e.g., Castonguay et al., 1995; Guy et al., 1992; Levitt,1980), but allow us, concomitantly with the cessation of stemelongation and the acquisition of freezing tolerance, to dem-onstrate the physiological consequences of cold exposure inpoplar.

Although sucrose, glucose and fructose are probably the moststudied soluble sugars in response to cold, raffinose and treha-lose present a higher correlation coefficient with the increasein freezing tolerance (Pearson’s coefficient: – 0.96 and – 0.76,

Spot #

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Fig. 5 Relative abundance of identified pro-teins (IOD: integrated optical density) appear-ing, or with increased expression, at 4 �C ascompared to the controls (23�C, 14 days).Proteins were classified following their func-tion and their role in stress responses. Fiveclusters are proposed: proteins showing chap-erone activity (a), drought-responsive pro-teins (b), proteins involved in stress signal-ling and transduction (c), enzymes involvedin antioxidant pathways (d) and unclassifiedproteins (e).

Chilling Effects on Poplar Plant Biology 6 (2004) 87

respectively), indicating a putative role for these carbohy-drates in cold acclimation of poplar. Indeed, sucrose and tre-halose can stabilise membranes and proteins in the case of de-hydration under freezing conditions. The stabilisation of pro-teins is ensured by preferential exclusion of sucrose from theprotein surface (Kendrick et al., 1997) while trehalose may bebound to hydrophilic domains of the proteins preventing inter-and intra-protein hydrogen bonds (Leslie et al., 1995). Mem-brane stabilisation is ensured by the insertion of these disac-charides in the membranes (Oliver et al., 1998). Moreover, theaccumulation of raffinose may supplement sucrose in mem-brane stabilisation and might help to prevent sucrose crystal-lisation during the glassy state (Caffrey et al., 1988). Recently, ithas been suggested that glucose and trehalose have functionsin the regulation of growth and photosynthesis (Paul and Pell-ny, 2003). Carbohydrates, and more particularly polysacchar-ides like UDP-glucose, are precursors of cell wall polysacchar-ides. However, given the reduction of growth observed in pop-lar at 4 �C, new tissues are fewer, and it is possible that carbo-hydrates did not enter into the cycle of cell wall formation.

In order to determine the main pathways involved in cold ac-climation of poplars, proteome analyses were performed ondays 7 and 14 of cold exposure. Sixty proteins out of over 800leaf proteins examined (on a pH range of 4 to 6) were found tochange reproducibly in abundance in response to cold expo-sure. These proteins could be grouped into clusters based ontheir function (Salekdeh et al., 2002). All changes in abundancemay be due to protein synthesis and breakdown, but it cannotbe excluded that some changes reflected differential binding ofproteins to insoluble components. Similarly, we are also un-able to state whether the observed changes are reflections ofchanges in gene expression at the transcript level (Gygi et al.,1999).

Considering that the spots decrease in their expression or dis-appear at 4 �C, it appears that all of them are involved in theenergy metabolism, i.e., UTPG-1-phosphate uridylyltransfer-ase (spot 46), UDPG dehydrogenase (spot 41), ATP synthase

beta unit (spot 47), glucosyltransferase (spot 48) and a precur-sor of the Rubisco large subunit (spot 42). These observationsmay be related to elongation cessation (e.g., UDPG dehydro-genase) and the accumulation of the different carbohydratesand confirm that cold exposure induces cellular perturbationsleading to a redistribution of energy fluxes.

During the cold treatment, poplar improves its defencesagainst reactive oxygen species and other reactive compounds.Among other things, the scavenging of these reactive com-pounds is enhanced by an accumulation of detoxifying en-zymes, such as ascorbate peroxidase (spot 19), peroxiredoxin(spot 20) or thioredoxin (spot 37) in cold-exposed leaves, as re-vealed by 2DE analyses. Indeed, these enzymes can detoxifysome of the different reactive forms present in plant cells (Mit-tler, 2002; Rouhier et al., 2001). Similarly, cold treatment isknown to affect the water status of plants. In this context,against a putative dehydration occurring at 4 �C, poplar syn-thesizes new proteins. At least two of them were identifiedin 2DE processed gels: one acidic dehydrin (spot 21) and oneLEA (spot 60). Beside 2DE analyses, other dehydrins, as wellas stable protein1-like proteins, were detected by immuno-logical analyses during cold acclimation (results not shown).These proteins are responsive to water stress and involved inthe stabilisation of proteins in the membrane or in the matrix(Borovskii et al., 2002; Wang et al., 2002). However, LEAs andstable protein1-like proteins are not the only defensive pro-teins synthesized during the cold acclimation of poplar. In-deed, molecular chaperone-like proteins, including heatshock-like proteins (smallHSP, spot 24, 55; HSP70, spots 3, 4,6, 8, 40; fragment of HSP70, spot 62; heat shock cognate 70,spot 39) and chaperonin 60 (spot 7), were more abundant un-der stress conditions than at optimal growth conditions. Theelevated proportion of chaperonins produced at 4 �C (10 on 21identified spots) indicates that existing and newly synthesizedproteins require stabilisation of their structural conformationby molecular chaperones to be translocated and/or fulfil theirfunctions.

Cold acclimation is also a multifactorial phenomenon whichinvolves overlapping stress responses (cold, drought and oxi-dative stresses, as mentioned above) and, unsurprisingly, si-multaneous modifications in several signalling pathways,transduction and transcription networks, as confirmed by theidentification of MAPKs (spots 34, 59), annexin (spot 17) andCBF1 (spot 23) on 2DE gels. Cold stress is one of the most sig-nificant factors leading to decreases in crop yield. Mechanismsby which plants cope with environmental stress are essentialfor their survival and productivity and are the key to their de-velopment. The integration of metabolic and environmentalsignals is possible via a network that regulates gene expressionduring stress. This network presupposes the perception of theexternal stress signal, transduction and activation of gene ex-pression, with consequences in terms of cellular metabolism.Signal perception can occur as an alarm triggered by H2O2,ABA, Ca2+ or other fast response systems (Sung et al., 2003).All these molecules may activate a specific and/or general re-sponse via proteins, which leads to modifications in gene ex-pression (Arora and Wisniewski, 1995).

Proteomics links physiological observations and genomicknowledge of cold stress tolerance. Many studies are currentlyaddressing the regulation of the transcriptome under different

23°C

4°C–7d

4°C–14d

41 42 46

Spot #

47 48

0

200

400

Vo

lum

e(I

OD

)

600

Fig. 6 Relative abundance of identified proteins (IOD: integrated op-tical density) disappearing, or with a decreased expression, at 4 �C ascompared to the controls (23�C, 14 days). All proteins were involvedin energy metabolism.

Plant Biology 6 (2004) J. Renaut et al.88

stress conditions (Desikan et al., 2001; Fowler and Thoma-show, 2002). These studies highlight a large number of geneswhich are stress-regulated and the lack of knowledge of mech-anisms of regulation. However, mRNA levels do not necessarilycorrespond to protein levels. Analysis of the proteome in thesame conditions will provide new insights in these studies.However, this technique does not offer the same high through-put as microarrays, for example. Some bottlenecks are stillpresent, such as the identification of proteins by MALDI-TOF-MS or LC-MS-MS without a fully sequenced genome support-ing the analysis. For example, up to August 2003, only 55 pro-teins from Populus tremula × P. tremuloides were available inthe TrEMBL or Swiss-Prot databases. However, this technicalgap will soon be filled with the publication of the poplar ge-nome. The pooling of proteomics and mass spectrometry withphysiological and genetic analyses will probably be a key stepin understanding the control of stress tolerance.

In conclusion, the results presented here indicate that poplar isa fast-responsive and chilling-tolerant species. Combined withproteome analyses, poplarmay be used to complete the under-standing of the cold acclimation phenomenon,with the specialfeatures inherent in woody species. In particular, it was shownthat cold acclimation must be considered as a multigenic phe-nomenon that triggers a response network in relation to stressperception, direct effects of cold (structural and biochemicaleffects) and indirect effects (putative oxidative and waterstresses) and allows the plant to enhance its tolerance.

Acknowledgements

Part of this study could be carried out thanks to support fromthe Luxembourg Ministry of Finances (REGECON researchproject), the Luxembourg Ministry of Culture, Higher Educa-tion and Scientific Research (J. R.’s PhD fellowship, BFR 99-031), and the International Plant Genetic Resources Institute(IPGRI, Rome). This work has also been carried out in partwithin the framework of an EC funded project, ESTABLISH(contract #OLK5-CT-2000-01377). The authors wish to thankDr. Brunisholz (Institut Molekularbiologie und Biophysik, Zu-rich) for performing the MS analyses. The authors would liketo thank Mr G. Heinen for his support and Drs. Danièle Evers,Bart Panis and Daniel Reisen for their helpful comments onthe manuscript and Dr. Henry-Michel Cauchie for statisticalanalyses.

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J.-F. Hausman

CRP-Gabriel LippmannCREBS, 162a Av. de la Faïencerie1511 LuxembourgGD Luxembourg

E-mail: hausman@crpgl.lu

Section Editor: H. Rennenberg

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