Original article

Dehydrin induction during drought and osmotic stress inPopulusAurore Caruso, Domenico Morabito, Francis Delmotte, Guy Kahlem, Sabine Carpin *

Laboratoire de biologie des ligneux et des grandes cultures, UFR, faculté des Sciences, université d’Orléans (UPRES EA 1207),rue de Chartres BP 6759, 45067 Orléans cedex 2, France

Received 23 May 2002; accepted 28 June 2002

Abstract

A cDNA encoding a dehydrin prepared from poplar (Populus euramericana cv Dorskamp clone) leaf mRNA was characterized. ThecDNA, designatedpeudhn1, is 1046 nucleotides long and has a 675 bp open reading frame with a 225 residues deduced amino acidsequence. The Peudhn1 sequence contains two conserved repeats of the characteristic lysine-rich-K-segment (EKKGIMDKIKEKLPG)preceded by an 8-serine residue stretch, but the Y-segment (DEYGNP) conserved motif, common to many dehydrins, is absent. Thesequence of the predicted Peudhn1 protein shows structural domains similar toArabidopsis thaliana ERD14, COR47 and LTI29 dehydrins.In unstressed poplar cuttings, a low level ofpeudhn1 transcripts was clearly detected in leaf and root tissues.Peudhn1 was induced bywithholding water, salt stress, cold and osmotic stress generated by addition of PEG 6000. A precocious induction ofpeudhn1 transcript inleaves was observed within 5 min of PEG 6000 treatment in hydroponic condition. © 2002 Éditions scientifiques et médicales ElsevierSAS. All rights reserved.

Keywords: Dehydrin; Hydroponic culture; Osmotic stress;Populus euramericana; Water stress

1. Introduction

Plants must possess and maintain adaptive mechanismsto ensure survival during periods of adverse environmentalconditions. Salt stress, freezing and drought stress are themost severe environmental conditions, affecting particularlyplant growth, crops yields and wood production. Bothdrought stress and salinity lead to a reduction of waterpotential in the root zone and make water acquisition byplants difficult [2]. Plants respond to this dehydrationthrough modifications of their morphology and physiologi-cal processes. The most studied modifications in response todehydration are stomatal closure[13], osmotic adjustment[23] and up-regulation gene expression[24]. One commonapproach to elucidate the molecular mechanisms of stresstolerance in plants is to identify and characterize genes

induced under these adverse environmental conditions.Studies were usually performed on herbaceous plants suchas Arabidopsis thaliana or tomato. It seems important todetermine if woody plants show the same molecular re-sponse to drought stress. Its economic importance, rela-tively small genome and potential for transformation byagrobacterium make poplar a good tree model.

Many dehydration induced proteins are referred to as lateembryogenesis abundant (LEA) proteins[24]. Dehydrins,also called LEA D11-family group II or RAB (responsive toABA), accumulate in plant tissues in response to cellulardehydration resulting from developmental events or envi-ronmental stimuli such as osmotic stress and low tempera-ture but also abscisic acid treatment[6,9,11,16,18,30]. Close[9,10] has reviewed recent progress in the mapping ofdehydrin genes and pointed out that these proteins are keycomponents of dehydration tolerance. It is hypothesized thatLEA proteins bind water in their random coil conformationand protect cellular structures from dehydration stress[24].Many studies indicate that dehydrins are associated withmacromolecules such as nucleoprotein complexes in thenucleus[20] and endomembranes in the cytoplasm[36]. Animmuno-localization study in wheat showed that dehydrinsare associated with the plasma membrane[19].

Abbreviations: ABA, abscisic acid; LEA, late embryogenesis abundant;PEG, polyethylene glycol; RAB, responsive to ABA; RACE, rapidamplification complementary end; RT-PCR, reverse transcription-polymerase chain reaction; RWC, relative water content

* Corresponding author.E-mail address: sabine.carpin@univ-orleans.fr (S. Carpin).

Plant Physiol. Biochem. 40 (2002) 1033–1042

www.elsevier.com/locate/plaphy

© 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved.PII: S 0 9 8 1 - 9 4 2 8 ( 0 2 ) 0 1 4 6 8 - 7

Using specific antibodies, Sauter et al. [35] have shownthe existence of dehydrins in protein extracts of Populusnigra and P. deltoides. The aim of this work was to analyzedehydrin gene expression in a P. euramericana(P. deltoides × P. nigra) Dorskamp clone under dehydrationconditions. Experimental drought stress is mainly obtainedby withholding water. A similar effect can also be producedby addition of a non-permeant osmolyte such as PEG 6000to the culture medium [6], or alternatively, a permeant onesuch as NaCl [27]. In the present study, different experi-mental conditions known for their effect on dehydrin geneinduction were tested: (a) drought stress obtained either bywithholding water or by adding PEG 6000 (non-permeantosmolyte) or NaCl which both mimic water deficit; (b) coldstress.

2. Results

2.1. Peudhn1 characterization

Application of the RT-PCR method using primers (P2and P3) designed from the amino acids consensus sequenceDKIKEKLPG [5,9] allowed isolation of two partial cDNAs.

After cloning and sequencing, these two cDNAs ap-peared to correspond to dehydrin genes. These clones werecalled peudhn1 and peudhn2. Complete sequence of peu-dhn1 cDNA, which is different from peudhn2, was obtainedby RACE-PCR. The resulting 1046 bp peudhn1 cDNA(Fig. 1) has a coding sequence of 675 bases correspondingto 225 amino acids with a deduced molecular mass of25,500 Da and a calculated isoelectric point of 5.17. ThiscDNA is characterized by two dehydrins specific sequences(KIKEK(L/I)PG), as well as a group of eight serine residuesand thus belongs to the SKn dehydrin group [9,10]. Thisdehydrin contains neither the Y (DEYGNP) segment nor anN-terminal peptide signal and is deficient in cysteine andtryptophane residues. An N-glycosylation potential site, 10phosphorylation sites as well as two myristoylation sites areidentified. This peudhn1 cDNA presents a high homologywith the nucleic acid and amino acid sequences of otherdehydrins, and a particularly high identity (46–48%) withthree A. thaliana dehydrins COR47, ERD14 and LTI29(Fig. 2).

Peudnh1 cDNA contains one EcoRV restriction site butno site for EcoRI or BamHI. Total DNA of P. euramericanaDorskamp clone was digested with EcoRI, BamHI, EcoRV,EcoRI–EcoRV and BamHI–EcoRV and screened with peu-dhn1 probe. Using highly stringent conditions, two EcoRIand two BamHI restriction fragments hybridized strongly,suggesting the existence of two peudhn1 structural genes.Further evidence was provided by digesting total DNA withdifferent combinations of the three restriction enzymes(Fig. 3).

2.2. Physiological and molecular effects of withholdingwater treatment

Withholding water did not induce significant modifica-tion of predawn water potential (Ww) since it remainedat –0.5 MPa during the first 7 d (Fig. 4A). From day 10, Wwdecreased significantly and reached –1.5 MPa on day 11.After re-watering Ww returned to its initial value within 1 d(Fig. 4A). The change in relative water content (RWC)followed that of the Ww (Fig. 4B). Osmotic potential (Wπ)increased from day 5 until day 11 and decreased whencuttings were re-watered (Fig. 4C). The leaf area increasedapproximately 30 cm2 d–1 per cuttings during the first 7days (Fig. 4D). Between days 7 and 10 the rate of leafgrowth decreased 3-fold (10 cm2 d–1). Leaf growth ceasedbeyond day 10. Re-watering induced a rapid resumption ofleaf growth, which corresponded to 15 cm2 d–1 at the end ofthe treatment. On day 0, stomatal conductance (g) was225 mmol H2O m–2 s–1 (Fig. 4E). Withholding water did notinduce a significant g variation until day 8. From days 8–11,a four-fold g decrease was observed. Re-watering of cut-tings allowed a stomatal reopening which became signifi-cant after 3 d.

Under control conditions (day 0) peudhn1 transcriptswere detected in leaves (Fig. 5). The transcript levelincreased during withholding water to reach a maximum onday 10 after which it decreased but remained highercompared to control. After 24 h of re-watering the transcriptlevel was similar to the one observed after 11 d of droughtstress. Three days after re-watering, transcript reached ahigher level than the one observed after 10 d of water deficit(Fig. 5).

2.3. Physiological and molecular effects of NaCland PEG 6000 treatments

During the whole experiment the Ww value remainedaround –0.4 MPa in control leaves (Fig. 6A). PEG 6000 andNaCl treatments induced a progressive Ww decrease, at –0.7and –0.85 MPa, respectively, after 23 d. For both stresses, asmall Ww decrease was observed after 1 d. For NaCl treatedcuttings, a progressive Ww decrease occurred until day 3reaching –0.7 MPa. Between days 1 and 3 the PEG 6000treated cuttings Ww returned to control value at –0.4 MPa.For NaCl treated cuttings, a Ww increase was observed fromdays 3–7. Afterward, in both treatments, a progressive andlinear Ww decrease was observed. This decrease was moresignificant under NaCl stress. RWC of leaves was notsignificantly different in control and NaCl treated cuttingsbut it decreased steadily in PEG 6000 treated cuttings (Fig.6B). A small Wπ increase was observed from day 14 forboth treatments (Fig. 6C). Leaf growth remained low untilday 8 in the three conditions (Fig. 6D). The rate of leafgrowth then increased to reach 25 cm2 d–1 in the control andNaCl treated cuttings, but only 17 cm2 d–1 in PEG 6000treated cuttings. Stomatal conductance was more reduced

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with PEG 6000 than with NaCl treatment (Fig. 6E). After23 d of treatment, leaves of stress cuttings exhibited someyellowing. This effect was more noticeable under PEG 6000treatment particularly on the oldest leaves (Fig. 7). Peudhn1transcript accumulation increased after NaCl and PEG 6000treatments (Fig. 8). On day 3, intense signals were observedfor both treatments with the strongest signal for NaCltreated cuttings based on the peudhn1 transcript levelcompared to the ribosomal level. For both stress conditions,a significant peudhn1 transcript decrease was observed onday 7, even when the rRNA control remained constant. Themaximum transcript level was reached 23 d after the begin-

ning of the treatments with a slightly stronger signal forNaCl treated cuttings. After 10 d of withholding water(Fig. 4A), which induced a Ww of –0.8 MPa, the accumu-lation of peudhn1 transcripts (Fig. 5) was higher than theone observed for the same Ww after 23 d of NaCl orPEG 6000 treatments (Figs. 6A–8).

2.4. Physiological and molecular effects of PEG 6000treatment under hydroponic condition

PEG 6000 addition to the hydroponic nutrient solutionbrought about a fast osmotic shock, resulting in a significant

Fig. 1. Complete nucleotide and deduced amino acid sequence of Peudhn1 (accession no. AJ300524). The three primers used to produce the full cDNA areshown (P2, P3, P4). The codon stop is marked with an asterisk. The first box designates the group of eight serine residues, and the two other boxes designatea specific dehydrin sequence.

A. Caruso et al. / Plant Physiol. Biochem. 40 (2002) 1033–1042 1035

stomatal conductance (g) decrease during the 2 h stress(Fig. 9). During the first 20 min, g value stayed constant(250 mmol H2O m–2 s–1). Then, g decreased steadily toreach 60 mmol H2O m–2 s–1 after 2 h treatment, correspond-ing to a decrease of 75% with respect to the control.

Peudhn1 transcript level increased in leaves as well as inroots concomitantly with the stomatal conductance decrease

(Fig. 10A,B). As early as 5 min after the beginning of thetreatment an important increase of the peudhn1 transcriptswas observed in leaves, reaching a maximum level after60 min (Fig. 10A). The signal decreased between 60 and105 min and then increased until 120 min of treatment. Inroots, a rapid stimulation of peudhn1 transcripts was alsoobserved within 20 min of treatment (Fig. 10B). Afterward,peudhn1 transcripts increased gradually to a maximum after120 min. The peudhn1 gene appeared to be more weaklyexpressed in roots than in leaves. The maximum peudhn1expression induced by PEG 6000 in hydroponic conditionswas lower than the effect induced by withholding water(Fig. 5), but higher than those obtained in cuttings growingin pots and treated with PEG 6000 or NaCl (Fig. 8).

2.5. Physiological and molecular effects of lowtemperature treatment

Low temperature stress (10 °C) led at once to a stomatalconductance decrease (Fig. 11) until an almost total sto-matal closure after 4 d (25 mmol H2O m–2 s–1). A significantincrease of the peudhn1 gene expression was observed onday 7 then its level fell to control levels on day 15 (Fig. 12).

Fig. 2. Sequence alignment created with the Clustal program (Infobiogen) show the conserved sequences between Peudhn1 of P. euramericana cloneDorskamp and three dehydrins of A. thaliana.

Fig. 3. Southern blot analysis of P. euramericana genomic DNA digestedwith different restriction enzymes: (a) EcoRI, (b) EcoRV, (c) BamHI, (d)EcoRI-EcoRV, (e) BamHI–EcoRV and hybridized with peudhn1 probe.

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Fig. 4. Time course of predawn leaf water potential (A), RWC (B), osmoticpotential (C), leaf surface increment (D) and stomatal conductance (E) ofP. euramericana clone Dorskamp submitted to drought stress by withhol-ding water. Vertical bars represent S.E. values. Means (A–E) werecompared to the value obtained at day 0, for (D) the means obtained afterre-watering were compared to day 11; (*) significant differences at P < 0.05(ANOVA test).

Fig. 5. Detection of mRNA encoding Peudhn1 in leaves of P. eurameri-cana clone Dorskamp submitted to drought stress by withholding waterfrom 0 to 11 d and re-watering from +1 to +3 d. Northern analysis wasperformed with 20 µg of total RNA. The detection of ribosomal 18S RNAwas performed as a control.

Fig. 6. Time course of predawn leaf water potential (A), RWC (B), osmoticpotential (C), leaf surface increment (D) and stomatal conductance (E) ofP. euramericana clone Dorskamp submitted to control conditions (¶) oriso-osmotic levels of NaCl (N) or PEG 6000 (▲). Vertical bars representS.E. values. Means were compared between control and stressed cuttings ateach date of measurement; (*) significant differences at P < 0.05 (ANOVAtest).

A. Caruso et al. / Plant Physiol. Biochem. 40 (2002) 1033–1042 1037

Fig. 7. Chlorose symptoms observed on old leaves (O) and young leaves (Y) of P. euramericana clone Dorskamp submitted to 23 d of NaCl (3), PEG 6000(2) or control conditions (1).

Fig. 8. Detection of mRNA encoding Peudhn1 in leaves of P. eurameri-cana clone Dorskamp submitted to control (C), NaCl (N) or PEG 6000treatments (P) from 1 to 23 d. Northern analysis was performed with 20 µgof total RNA. The detection of ribosomal 18S RNA was performed as acontrol.

Fig. 9. Time course of stomatal conductance of P. euramericana cloneDorskamp submitted to drought stress induced by PEG 6000 (50 g l–1)under hydroponic conditions. Vertical bars represent S.E. values; (*)significant differences at P < 0.05 (ANOVA test).

Fig. 10. Detection of mRNA encoding Peudhn1 in leaves (A) and roots (B)of P. euramericana clone Dorskamp submitted under hydroponic condi-tions to PEG 6000 (50 g l–1) from 0 to 120 min. Northern analysis wasperformed with 20 µg of total RNA. The detection of ribosomal 18S RNAwas performed as a control.

Fig. 11. Time course of stomatal conductance of P. euramericana cloneDorskamp submitted to drought stress induced by low temperature (10 °C).Means were compared between day 0 which correspond to control andstressed cuttings at each date of measurement; (*) significant differences atP < 0.05 (ANOVA test).

1038 A. Caruso et al. / Plant Physiol. Biochem. 40 (2002) 1033–1042

3. Discussion

In P. euramericana cv Dorskamp, resulting fromP. deltoides × P. nigra crossing, evidence for two dehydringenes, peudhn1 and peudhn2, was presented. In woodyplants, dehydrins have also been observed in angiospermssuch as lemon tree [22], peach tree [1] and birch [33] and ingymnosperms including spruce [32] and douglas fir [25].The peudhn1 cDNA was cloned and sequenced. Southernblot analysis revealed two EcoRI and two BamHI restrictionfragments of total poplar DNA hybridized with the peudhn1probe. The P. euramericana cv Dorskamp genome appearsto contain two homologous genes which could be allelesfrom both parents. However, only one peudhn1 transcriptsignal was detected by hybridization with the peudhn1probe. Peudhn1 dehydrin belongs to the SKn group andpresents, respectively, 48%, 47% and 46% identity withERD14, COR47 and LTI29 of A. thaliana [29]. The peu-dhn1 cDNA was obtained from an mRNA populationisolated from leaves of poplar cultivated under controlconditions. Therefore, the peudhn1 gene is constitutivelyexpressed as is Arabidopsis ERD14, as well as COR47 andLTI29, which are weakly expressed in leaves under controlconditions [29]. The constitutive peudhn1 gene expressionin P. euramericana Dorskamp clone could be associatedwith its drought tolerance [3,21]. A constitutive expressionof dehydrin genes was also observed in other woody plantorgans, such as spruce stems [32] or lemon tree leaves [22].In control conditions the Peudhn1 gene was more highlyexpressed in leaves than in roots. The same phenomenonwas observed for spruce [32], unlike the ArabidopsisERD14 gene which is more highly expressed in roots. Theexpression of dehydrin genes belonging to the SKn groupappears to be constitutive but the level is dependant onorgan and plant species. It is likely that, in absence of anypeptide signal, Peudhn1 has a cytoplasmic localization.However, the potential phosphorylation of its serine resi-dues could allow a nuclear localization [19,20,26] where itcould have a protective function on macromolecules [10]and in particular on transcriptional mechanisms. Acidicdehydrin accumulation near plasma membranes could havea protective function as observed for WCOR410 wheatdehydrin which could be involved in the prevention ofplasma membrane destabilization occurring duringPEG 8000 or cold dehydration conditions [14]. The mecha-nism of the interaction of this dehydrin with the plasma

membrane surface remains unknown. It was proposed thatthe protein could avoid the damage caused by precipitationor crystallization of compounds whose intracellular concen-tration increases during dehydration [14].

The application of various stresses such as withholdingwater or addition of PEG 6000 or NaCl to soil resulted in aprogressive decrease of the predawn leaf water potential, adecrease of stomatal conductance and a growth reductionassociated with a progressive increase of Peudhn1 transcriptlevel. Rehydration of P. euramericana cv Dorskamp cut-tings after a withholding water period induces a newpeudhn1 transcript accumulation. This phenomenon is cor-related to water potential, stomatal conductance and leafgrowth restoration. Theses data differ from results observedwith sunflower or spinach where HaDhn1 [18] and CAP 85[28] transcripts either decreased or disappeared after re-hydration. These differences may be explained by the factthat the authors did not give any data relative to a possiblegrowth recovery after the stress. The increase of peudhn1transcripts could be explained by the recovery of transcrip-tional activity necessary for growth renewal. This dehydrinaccumulation could also give a better protection to growingorgans and allow hardness against a later dehydrationtreatment.

Unlike PEG 6000 treatment, leaf growth of NaCl treatedcuttings remained equal to control in spite of a lowerstomatal conductance which seems to be sufficient tomaintain a photosynthetic activity. Osmotic potential in-crease, observed during PEG 6000 treatment, is due to a fallof RWC while, for NaCl treatment, the osmotic potentialincrease could results from Na+ or Cl– absorption and fromthe synthesis of compatible compounds. In spite of amaintained leaf growth equal to control, Peudhn1 was morestrongly expressed in NaCl treated cuttings. This geneexpression seems correlated to the increase of osmoticpotential, mainly at the end of the stress period. The relativedecrease of peudhn1 transcripts occurring on day7 of NaClor PEG 6000 treatments seems to be associated with adecrease of stomatal conductance which could correspondto a transient decrease in metabolism.

Addition of 50 g l–1 of PEG 6000 in hydroponic condi-tions caused a rapid reduction of stomatal conductance.Root peudhn1 transcripts accumulated gradually to a maxi-mum level reached after 2 h. An important peudhn1 tran-script accumulation occurred in leaves compared to roots asearly as 5 min after treatment. The stress perception andsignal transduction which led to the positive regulation ofpeudhn1 transcription anticipated the stomatal closure. It isthe first time that such a rapid kinetic of any dehydrin geneexpression was observed. The rapid accumulation of peu-dhn1 transcripts in stressed leaves is unlikely to be theconsequence of intervention of a root secondary messagesuch as abscisic acid, well known to induce stomatal closure[12]. Since peudhn1 transcripts were detected after 5 min ofPEG 6000 treatment, leaf cells could perceive dehydrationdirectly due to root water absorption deficit and leaf

Fig. 12. Detection of mRNA encoding Peudhn1 in leaves of P. eurameri-cana clone Dorskamp submitted to low temperature (10 °C) from 1 to 15 d.Northern analysis was performed with 20 µg of total RNA. The detectionof ribosomal 18S RNA was performed as a control.

A. Caruso et al. / Plant Physiol. Biochem. 40 (2002) 1033–1042 1039

transpiration maintenance. Drought induction of genes inexcised tomato leaves [12] also demonstrates that theirtranscription can be independent from a root signal. Stresssignal perception could be carried out by a membraneosmosensor such as A. thaliana ATHK1 [39].

Numerous genes activated by dehydration stress havebeen shown to respond also to other biotic or abiotic stresses[3]. A low temperature treatment of P. euramericana cvDorskamp led to a reduction in stomatal conductance and alarge peudhn1 transcript accumulation as early as the firstday. Induction of dehydrin synthesis by cold has alreadybeen observed for numerous plants [14,22,32]. Nylander etal. [29] compared the levels of the cold induced COR47 andLTI29 dehydrin transcripts which demonstrate an accumu-lation kinetic similar to that observed for peudhn1.

The protective function of dehydrins remains hypotheti-cal, Cellier et al. [7] showed a good correlation betweendrought adaptation of sunflower and dehydrin accumula-tion. Since peudhn1 expression was stimulated by differentenvironmental stresses (low water potential, high ionicconcentration, low temperature), it could be involved instress tolerance. The isolation of the peudhn1 gene willallow the analysis of promoter consensus sequences in-volved in its regulation by different stress signals. Peudhn1gene expression in transgenic poplar could allow confirma-tion of the function of this protein.

4. Methods

4.1. Plant material and treatments

In order to study the induction of poplar dehydrins bydrought stress, we have chosen the clone P. euramericana[P. deltoides (Batr.) Marsh × P. nigra L.] cv Dorskamp(male) obtained at the Dorskamp Institut of Wageningen(Netherlands, 1952) and selected for its drought tolerance[4,21,38]. Nodal segments, obtained from trees grownoutdoors were excised and rooted in 1 l individual pots filledwith peat. These cuttings were watered before treatment tofield capacity and placed in a growth chamber undercontrolled environmental conditions. Temperature was21 ± 1 °C and 70% relative humidity (night and day), and anirradiance of 134 µmol s–1 m–2 was provided during16 h d–1 by fluorescent light. Two kinds of drought stresswere applied to the cuttings. (a) Progressive drought stresswas applied on 45-d-old cuttings (beginning on day 0 of thestress period) growing in pots and measuring 20 cm inlength. Treatments were carried out by withholding waterfor 11 d or by adding PEG 6000 (50 g l–1) or NaCl(1.36 g l–1) to the nutrient solution for 23 d. These solutionscorresponding to an osmotic potential of 43 mosmoles kg–1

of water (micro-osmometer, Roebling, Messtechnick) weresupplied three times per week with an amount of complete

nutritive solution equal to transpiration loss for PEG 6000(purum, Buch, Switzerland) and NaCl treatments. For thewithholding water experiment, re-watering was applied tonine water deprived cuttings by adding nutrient solution tofield capacity two days after that the predawn water poten-tial had reached a value significantly different to the control(–1.5 MPa). Low temperature treatment was performed on30 cuttings for 15 d in a growth chamber maintained at10 ± 1 °C and 70% relative humidity during night and day,and an irradiance of 134 µmol s–1 m–2 was provided during16 h per day by fluorescent light. (b) Rapid drought stresswas applied on 1-month-old rooted cuttings grown underhydroponic condition by supplementing the medium withPEG 6000 (50 g l–1) during 2 h. This method makes rootsampling easier and avoids any wounding stress effectoccurring during their harvesting [32]. Both control andtreated leaves were sampled periodically as shown inresults, frozen in liquid nitrogen and stored at –80 °C untiluse. Root analysis was performed only under hydroponicconditions.

4.2. Growth and water status measurements

During each progressive drought treatment, plant growthwas estimated by measuring the total leaf surface area ofnine cuttings as described by Brignolas et al. [4]. In order toestimate the water potential of soil in contact with roots, thepredawn leaf water potential (Ww) was measured on sixcuttings with a Scholander pressure chamber on two fullyexpanded leaves per plant. Stomatal conductance (g) wasperformed on nine cuttings and determined 6 h after thebeginning of the light period on both faces of two matureleaves with a steady state porometer (PMR-3, PP-Systems,GB). Results were calculated as gabaxial + gadaxial. Osmoticpotential (Wπ) was measured with an osmometer on twoleaves from six cuttings after they were frozen in dry ice andthawed. The leaf dry weight was determined on two leavescollected from six cuttings by drying at 105 °C for 3 h. TheRWC was calculated according to the following relation[40]: RWC (%) = (fresh weight – dry weight) 100/(saturatedweight – dry weight).

4.3. Nucleic acid purification

Whole leaves or roots were collected and pooled fromthree cuttings at sampling dates. Total RNAs were extractedaccording to Dean et al. [15] using the extraction bufferdescribed by Prescott and Martin [31] and modified byadding 2% (w/v) of polyvinylpyrrolidone (PVP 40000,UCB Leuven, Belgium) [8]. Poly(A+) RNAs were purifiedfrom 750 µg of total RNAs by three runs through a columnof oligodT cellulose (Oligotex, Qiagen, Hilden, Germany).

1040 A. Caruso et al. / Plant Physiol. Biochem. 40 (2002) 1033–1042

4.4. 3' race-PCR

The 3' cDNA fragments for dehydrin genes were ob-tained by the following RT-PCR procedure. ComplementaryDNA was prepared from 150 ng total RNAs in a reactionvolume of 60 µl according to the protocol of SenscriptReverse Transcriptase kit (Qiagen, Courtaboeuf, France).Modified oligodT16VN primer (P1) was used. Sequences ofnested primers 2 (P2) and 3 (P3), respectively, (5'-GARAARATHAARGARAARYTNCC-3' and 5'-ATHA-ARGARAARYTNCNG-3'; R = A/G; H = A/T/C; Y = C/T;N = A/C/T/G) correspond to the sense orientation ofDKIKEKLPG sequence (Fig. 1). PCR mediated amplifica-tion of the first strand cDNA synthesis products wasperformed with 0.2 µM of P1 and P2 and 2.5 units ofThermus aquaticus (Taq) DNA polymerase (Pharmacia-Amersham, Orsay, France). Thirty cycles of 30 s denatur-ation at 94 °C, 1 min annealing at 54 °C and 1 min exten-sion at 72 °C were performed. Amplified cDNA productswere submitted to a second PCR with P1 and P3 using thesame PCR conditions except with 35 cycles and except adecrease of P1 concentration (0.1 µM). Reactions werecompleted with a 10 min extension at 72 °C. PCR productswere cloned into pCR 2.1 TOPO vector (TA TOPO Cloningkit, Invitrogen, Groningen, The Netherlands). The ligationproducts were transformed into Escherichia coli TOP10.Recombinant bacteria were selected for further analysis.

4.5. Library construction and full peudhn1 cDNA cloning

cDNA were prepared using polyA+ RNAs obtained fromcontrol leaf tissues. Marathon cDNA Amplification Kit(Clontech, Palo Alto, CA, USA) was used to construct acDNA library. The amplification of the full peudhn1 cDNAwas achieved according to the manufacturer’s instructions.Two primers were used: the specific sequence peudhn1,primer 4 (P4): 5'GGGTCTAAAAAGGGGTCAAAA-TAAC-3' and one primer AP1 provided in the Race kit. Tocomplete amplification, 0.05 µM AP1 and 0.2 µM P4 wereused. PCR fragments were cloned in pCR 2.1 vector andsequenced on both strands (Eurogentec, Seraing, Belgium).

4.6. Northern blot analysis

Equal amounts of total RNAs samples (20 µg) weredenatured and fractionated by electrophoresis in a 1.2%agarose denaturing gel [34]. Total RNA quality was con-firmed by ribosomal RNA integrity observed after ethydiumbromide treatment. Gels were blotted by a capillary proce-dure [34] onto NY Plus membrane (Porablot, Macherey-Nagel, Düren, Germany). Fractionated RNAs were cross-linked at 80 °C. The radiolabeled peudhn1 probe wasobtained using Prime a Gene Labeling System kit (Promega,Madisson, WI, USA) with the cloned cDNA adding 50 µCi(330 nM) of [α32 P]dCTP. The probe was purified on G50micro-columns (Amersham-Pharmacia, Orsay, France).

Membranes were prehybridized in hybridization buffer [34]for 2 h prior adding [α32 P] dCTP radiolabeled probes1 × 1010 cpm µg–1. The ribosomal probe was synthesized byPCR using 200 ng of Dorskamp genomic DNA with theprimers NS50 (5'-GGGGGAGTATGGTCGCAAGGC-3')and NS61 (5'-TCAGTGTAGCGCGCGTGCGGC-3') [37].Membranes were exposed to X-ray film (Kodak) for 12 hat –70 °C. These experiments were replicated in triplicate.

4.7. Southern blot analysis

P. euramericana cv Dorskamp genomic DNA was ex-tracted from 2 g of leaves as described by Doyle and Doyle[17]. Genomic DNA was dissolved in 500 µl of sterile waterand treated by RNase. Sample was digested with therestriction enzymes EcoRI, BamHI and EcoRV. Digestionproducts were fractionated by electrophoresis on 0.8%agarose gel and transferred on an NY Plus membrane.Hybridization was performed at 60 °C with the peudhn1probe.

Acknowledgements

In memory to Professor Gilles Guerrier. The skillfultechnical assistance of Françoise Chefdor and RemiBénardeau is gratefully acknowledged. We are grateful toDr. Christiane Depierreux, Dr. Robert Hancock and Dr.Claude Penel for reading the manuscript, Dr. Philippe Labelfor useful technical advice.

References

[1] T.S. Artlip, A.M. Callahan, C.L. Bassett, M.E. Wisniewski, Seasonalexpression of a dehydrin gene in sibling deciduous and evergreengenotypes of peach (Prunus persica [L.] Batsch), Plant Mol. Biol.33 (1997) 61–70.

[2] H.J. Bohnert, E. Sheveleva, Plant stress adaptations-making metabo-lism move, Curr. Opin. Plant Biol. 1 (1998) 267–274.

[3] E.A. Bray, Plant responses to water deficit, Trends Plant Sci. 2(1997) 48–54.

[4] F. Brignolas, C. Thierry, G. Guerrier, E. Boudouresque, Comparedwater deficit response of two Populus euramericana clones, LuisaAvanzo and Dorskamp, Ann. Sci. Forest 57 (2000) 261–266.

[5] A. Campalans, M. Pagès, R. Messeguer, Protein analysis duringalmond embryo development. Identification and characterization ofa late embryogenesis abundant protein, Plant Physiol. Biochem. 38(2000) 449–457.

[6] N. Carpita, D. Sabularse, D. Montezinos, D.P. Delmer, Determina-tion of pore size of cell walls of living plant cells, Science 205(1979) 1144–1147.

[7] F. Cellier, G. Conéjéro, J.C. Breitler, F. Casse, Molecular andphysiological responses to water deficit in drought-tolerant anddrought-sensitive lines of sunflower. Accumulation of dehydrintranscripts correlates with tolerance, Plant Physiol. 116 (1998)319–328.

[8] S. Chang, J. Puryear, J. Cairney, A simple and efficient method forisolating RNA from pine trees, Plant Mol. Biol. Rep. 11 (1993)113–116.

A. Caruso et al. / Plant Physiol. Biochem. 40 (2002) 1033–1042 1041

[9] T.J. Close, Dehydrins: emergence of a biochemical role of a familyof plant dehydration proteins, Physiol. Plant. 97 (1996) 795–803.

[10] T.J. Close, Dehydrins: a commonalty in the response of plants todehydration and low temperature, Physiol. Plant. 100 (1997)291–296.

[11] T.J. Close, P.J. Lammers, An osmotic stress protein of cyanobacteriais immunologically related to plant dehydrins, Plant Physiol. 101(1993) 773–779.

[12] A. Cohen, E.A. Bray, Characterization of three mRNAs that accu-mulate in wilted tomato leaves in response to elevated levels ofendogenous abscisic acid, Planta 181 (1990) 27–33.

[13] G. Cornic, Drought stress inhibits photosynthesis by decreasingstomatal aperture – not by affecting ATP synthesis, Trends Plant Sci.5 (2000) 187–188.

[14] J. Danyluk, A. Perron, M. Houde, A. Limin, B. Fowler, N. Benha-moun, F. Sarhan, Accumulation of an acidic dehydrin in the vicinityof the plasma membrane during cold acclimation of wheat, PlantCell 10 (1998) 623–638.

[15] C. Dean, P. Elzen, S. Tamaki, P. Duinsmuir, J. Bedbrook, Differen-tial expression of the eight genes of the petunia ribulose biphosphatecarboxylase small subunit multigene family, EMBO J. 5 (1985)3055–3061.

[16] M. Del Mar Parra, O. Del Pozo, R. Luna, J.A. Godoy, J.A. Pintor-Toro, Structure of the dehydrine tas14 gene of tomato and itsdevelopmental and environmental regulation in transgenic tobacco,Plant Mol. Biol. 32 (1996) 453–460.

[17] J.J. Doyle, J.L. Doyle, Isolation of plant DNA from fresh tissue,Focus 12 (1990) 13–15.

[18] T. Giordani, L. Natali, A. D’Ercole, C. Pugliesi, M. Fambrini,P. Vernieri, C. Vitagliano, A. Cavallini, Expression of a dehydringene during embryo development and drought stress in ABA-deficient mutants of sunflower (Helianthus annuus L, Plant Mol.Biol. 39 (1999) 739–748.

[19] A. Goday, A.B. Jensen, F.A. Culianez-Macià, M.M. Albà,M. Figueras, J. Serratosa, M. Torrent, M. Pagès, The maize abscisicacid-responsive protein Rab17 is located in the nucleus and interactswith nuclear localization signals, Plant Cell 6 (1994) 351–360.

[20] J.A. Godoy, R. Lunar, J.A. Pintor-Toro, Expression, tissue distribu-tion and subcellular localization of dehydrin TAS14 in salt-stressedtomato plants, Plant Mol. Biol. 26 (1994) 1921–1934.

[21] G. Guerrier, F. Brignolas, C. Thierry, M. Courtois, G. Kahlem,Organic solutes protect drought-tolerant poplar against reactiveoxygen species, J. Plant Physiol. 156 (1999) 93–99.

[22] M. Hara, Y. Wakasugi, Y. Ikoma, Y. Masamichi, K. Ogawa,T. Kuboi, cDNA sequence and expression of a cold-responsive genein citrus unshiu, Biosci. Biotechnol. Biochem. 63 (1999) 433–437.

[23] P.D. Hare, W.A. Cress, J. Van Staden, Dissecting the roles ofosmolyte accumulation during stress, Plant Cell Environ. 21 (1998)535–553.

[24] J. Ingram, D. Bartels, The molecular basis of dehydration tolerancein plants, Annu. Rev. Plant Physiol. Plant Mol. Biol. 47 (1996)377–403.

[25] S.B. Jarvis, M.A. Taylor, M.R. Mac Leod, H.V. Davies, Cloning andcharacterisation of the cDNA clones of three genes that a differen-

tially expressed during dormancy breakage in the seeds of Douglasfir (Pseudotsuga menziesii), J. Plant Physiol. 147 (1996) 559–566.

[26] A.B. Jensen, A. Goday, M. Figueras, A.C. Jessop, M. Pagès,Phophorylation mediates the nuclear targeting of the Maize RAB17protein, Plant J. 13 (1998) 691–697.

[27] J. Mexal, J.T. Fisher, J. Osteryoung, C.P.P. Reid, Oxygen availabilityin polyethylen glycol solutions and its implications in plant-waterrelations, Plant Physiol. 55 (1975) 20–24.

[28] L.G. Neven, D.W. Haskell, A. Hofig, Q.B. Li, C.L. Guy, Character-ization of a spinach gene responsive to low temperature and waterstress, Plant Mol. Biol. 21 (1993) 291–305.

[29] M. Nylander, J. Svensson, E.T. Palva, B.V. Welin, Stress-inducedaccumulation and tissue-specific localization of dehydrins in Arabi-dopsis thaliana, Plant Mol. Biol. 45 (2001) 263–279.

[30] O. Ouvrard, F. Cellier, K. Ferrare, D. Tousch, T. Lamaze, J.M. Du-puis, F. Casse-Delbard, Identification and expression of water stress-and abscisic acid-regulated genes in drought-tolerance sunflowergenotype, Plant Mol. Biol. 31 (1996) 819–829.

[31] A. Prescott, C. Martin, A rapid method for the quantitative assess-ment of leaves of specific mRNAs in plants, Plant Mol. Biol. Rep.4 (1987) 219–224.

[32] S. Richard, M.J. Morency, C. Drevet, L. Jouanin, A. Seguin,Isolation and characterization of a dehydrin gene from white spruceinduced upon wounding, drought and cold stresses, Plant Mol. Biol.43 (2000) 1–10.

[33] P.L.H. Rinne, P.L.M. Kaikuranta, L.H.W. Van der Plas,C. Van der Schoot, Dehydrins in cold-acclimated apices of birch(Betula pubescens Ehrh.): production, localization and potential rolein rescuing enzyme function during dehydration, Planta 209 (1999)377–388.

[34] J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular Cloning: Alaboratory Manual, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, NY, 1989.

[35] J.J. Sauter, S. Westphal, M. Wisniewski, Immunological identifica-tion of dehydrin-related proteins in the wood of five species ofPopulus and Salix caprea L, J. Plant Physiol. 154 (1999)781–788.

[36] K. Schneider, B. Wells, E. Schmelzer, F. Salamini, D. Bartels,Desiccation leads to the rapid accumulation of both cytosolic andchloroplastic proteins in the resurrection plant Craterostigma plan-tagineum Hochst, Planta 189 (1993) 120–131.

[37] L. Simon, M. Lalonde, T.D. Bruns, Specific amplification of 18Sfungal ribosomal genes from vesicular-arbuscular endomycorrhisalfungi colonising roots, Appl. Environ. Microbiol. 58 (1992)291–295.

[38] G. Soulères, Le point sur Dorskamp. Bull. vulgarisation forestière:Forêt Entreprise, IDF 54 (1988) 15-31.

[39] T. Urao, B. Yakubov, R. Satoh, K. Yamaguchi-Shinozaki, M. Seki,T. Hirayama, K. Shinozaki, A transmembrane hybrid-type histidinekinase in Arabidopsis functions as an osmosensor, Plant Cell 11(1999) 1743–1754.

[40] P.E. Weatherley, Some aspects of water relations, Adv. Bot. Res. 3(1970) 171–206.

1042 A. Caruso et al. / Plant Physiol. Biochem. 40 (2002) 1033–1042