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Tree Physiology 31, 1335–1355doi:10.1093/treephys/tpr083

Comparative study of transcriptional and physiological responses to salinity stress in two contrasting Populus alba L. genotypes

Isacco Beritognolo1,2, Antoine Harfouche1, Federico Brilli3, Gianluca Prosperini4, Muriel Gaudet1, Mikael Brosché5, Francesco Salani1, Elena Kuzminsky1, Petri Auvinen6, Lars Paulin6, Jaakko Kangasjärvi5, Francesco Loreto7, Riccardo Valentini1, Giuseppe Scarascia Mugnozza8 and Maurizio Sabatti1,9

1Department for Innovation in Biological, Agro-Food and Forest Systems (DIBAF), University of Tuscia, Via S. Camillo de Lellis, 01100 Viterbo, Italy; 2Present address: Institute for Agriculture and Forest Systems in the Mediterranean (CNR-ISAFOM), National Research Council, Via Madonna Alta 128, 06128 Perugia, Italy; 3Institute of Agro-environmental and Forest Biology (CNR-IBAF), National Research Council, Via Salaria Km 29,300, 00015 Monterotondo Scalo, Roma, Italy; 4CASPUR, Consorzio interuniversitario per le applicazioni di supercalcolo per università e ricerca, Via dei Tizii 6, 00185, Roma, Italy; 5Department of Biosciences, Division of Plant Biology, University of Helsinki, Viikinkaari 1 (PL 65) 00014 Helsinki, Finland; 6Institute of Biotechnology, University of Helsinki, 00014 Helsinki, Finland; 7Plant Protection Institute (CNR-IPP), National Research Council, Via Madonna del Piano, 10 50019 Sesto Fiorentino, Firenze, Italy; 8Department of Agronomy, Forestry and Land Use (DAF), Agricultural Research Council (CRA), Via del Caravita 7/a, 00186 Roma, Italy; 9Corresponding author (sabatti@unitus.it)

Received May 26, 2011; accepted July 27, 2011; published online September 12, 2011; handling Editor Roberto Tognetti

Soil salinity is an important limiting factor to tree growth and productivity. Populus alba L. is a moderately salt-tolerant spe-cies and its natural populations are adapted to contrasting environments, thus providing genetic resources to identify key genes for tolerance to abiotic stress, such as salinity. To elucidate the molecular and genetic basis of variation for salinity tolerance in P. alba, we analyzed the short-term ecophysiological and transcriptome response to salinity. Two contrasting genotypes, 6K3, salt sensitive, and 14P11, salt tolerant, originating from North and South Italy, respectively, were challenged with salt stress (200 mM NaCl). Sodium accumulated in the leaves of salt-treated plants and its concentration increased with time. The net photosynthesis was strongly reduced by salinity in both genotypes, with 6K3 being significantly more affected than 14P11. The transcriptional changes in leaves were analyzed using cDNA microarrays containing about 7000 stress-related poplar expressed sequence tags (EST). A microarray experiment based on RNA pooling showed a number of salinity- regulated transcripts that markedly increased from 3 h to 3 days of salinity treatment. Thus, a detailed analysis was performed on replicated plants collected at 3 days, when ~20% of transcripts showed significant change induced by salinity. In 6K3, there were more genes with decreased expression than genes with increased expression, whereas such a difference was not found in 14P11. Most transcripts with decreased expression were shared between the two genotypes, whereas transcripts with increased expression were mostly regulated in a genotype-specific manner. The commonly decreased transcripts (71 genes) were functionally related to carbohydrate metabolism, energy metabolism and photosynthesis. These biological pro-cesses were consistent with the strong inhibition of photosynthesis, caused by salinity. The commonly increased transcripts (13 genes) were functionally related to primary metabolism and biosynthesis of proteins and macromolecules. The salinity-increased transcripts discriminated the molecular response of the two genotypes. In 14P11, the 21 genes specifically salinity-induced were related to stress response, cell development, cell death and catabolism. In 6K3, the 15 genes with salinity-increased expression were involved in protein biosynthesis, metabolism of macromolecules and cell organization and biogenesis. The difference in transcriptome response between the two genotypes could address the molecular basis of intra-specific variation of salinity tolerance in P. alba.

Keywords: microarray, photosynthesis, salt stress, transcriptomics, white poplar.

Research paper: Part of a special section on poplars and the environment

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Introduction

Salinity is a widespread environmental constraint affecting plant biomass production and agricultural productivity. Given the growing agricultural water scarcity and increased area of salt-affected land, the ability of crop plants to tolerate high salt concentrations is now an agriculturally useful trait (Rus et al. 2005). A full understanding of the physiological and molecular foundations of salt tolerance may help us in producing plants that better cope with salinity. Most plant species are sensitive to high concentrations of Na+, which causes combined ion tox-icity and osmotic stress (Horie et al. 2009). Thus, plants have evolved intricate tolerance mechanisms for dealing with salt stress (Zhu 2002). This capacity has been described as a quantitative genetic trait (Rus et al. 2005). Therefore, due to the large number of genes involved in the response to salt stress, one way to dissect this complex trait is to use micro-arrays and monitor global gene expression changes in plants. Microarrays provide a combination of technical and practical advantages that make them the most widely used platform for transcriptional profiling (Deyholos 2010).

During the last decade, our understanding of the processes underlying plant response to salinity, at the molecular and whole-plant levels, has rapidly progressed. Salt tolerance is controlled by many genes involved in different pathways, such as ion exclusion, ion compartmentalization in vacuoles, ion selectivity, synthesis of compatible solutes and scavenging of reactive oxygen species (ROS) (Blumwald et al. 2000, Sakamoto and Murata 2002, Zhu 2003, Katiyar-Agarwal et al. 2006, Munns and Tester 2008). Notably, an essential compo-nent of plant salinity tolerance is the salt overly sensitive 1 (SOS1) protein, a sodium/proton antiporter that was originally identified in Arabidopsis (Shi et al. 2000) and has also been isolated from Populus euphratica Oliv. (Wu et al. 2007) and from Populus trichocarpa Torr. & A. Gray (Tang et al. 2010).

Tolerance and sensitivity to abiotic stress are unequally dis-tributed among plant species (Oh et al. 2010). In most families characterized by salt-sensitive species, salt-tolerant species can also be found. Hence, the selection of appropriate germplasm is of paramount importance in successfully improving salt toler-ance in plants. The use of stress-susceptible species (e.g., Arabidopsis thaliana) to study drought and salinity stress has been criticized owing to the limited defence mechanisms of these plants, and their response to stress may actually reflect death or senescence rather than useful adaptation (Munns and Tester 2008). Microarray-based comparison of variation between and within closely related species that differ in their tolerance to stress is a potentially powerful strategy for identifi-cation of inter- and intra-specific genetic variability in salt toler-ance. For example, microarrays have been increasingly used to analyze halophyte transcriptome in comparison to those of gly-cophytic relatives (Taji et al. 2004, Gong et al. 2005, Chelaifa

et al. 2010). Stress transcriptome studies have now also been reported in drought- or salinity-treated rice (Oryza sativa L.) (Walia et al. 2005), wheat (Triticum spp.) (Mohammadi et al. 2008, Ergen et al. 2009), sugarcane (Saccharum spp.) (Rodrigues et al. 2009), potato (Solanum tuberosum L.) (Mane et al. 2008), tomato (Solanum spp.) (Sun et al. 2010b) and poplar (Populus spp.) (Brosché et al. 2005, Berta et al. 2010, Brinker et al. 2010, Ding et al. 2010, Janz et al. 2010). Most of these studies take advantage of comparative transcriptomics, in which species or genotypes with different stress tolerance are compared. The Populus genus includes species of immense ecological and commercial importance, but with remarkable variability in salt tolerance. Populus euphratica, a species accli-mated to saline and hot environments, has been widely studied as a model plant and experimentally compared with less toler-ant poplar species to elucidate physiological and molecular mechanisms of salt tolerance in trees (Chen and Polle 2010). However, intra-species comparative studies may also provide additional important information on salt tolerance mechanisms in poplars. Populus alba L. (white poplar), one of the potentially more interesting woody plants for high biomass production, has considerable genetic variation for salt tolerance due to its natu-ral adaptation to dry environments. Because genetic variation is subjected to natural selection in contrasting ecological habitats, two P. alba genotypes, 14P11, a salt- tolerant genotype of south-ern origin, and 6K3, a salt-sensitive geno type of northern origin, were selected for this study. These genotypes have previously shown substantial phenotypic differences in their biomass pro-duction, environmental adaptation (Marron et al. 2011), toler-ance to soil salinity (Beritognolo et al. 2007, Abbruzzese et al. 2009) and response to cadmium (Cocozza et al. 2008).

The objective of this study was to compare the sensitive and tolerant genotypes of P. alba in order to identify genes that likely contribute to the intra-specific variation of salinity toler-ance. We compared the physiological responses and transcrip-tome regulation in leaves of these two genotypes after exposure to an acute soil salinity stress. The physiological and molecular analyses were focused on the functionality of the photosynthetic apparatus, which often limits growth and pro-duction in salt-stressed plants. The microarray-based compari-son of transcriptome profiles identified a number of genotype-specific genes and biological processes that are involved in intra-specific variation of salt tolerance in poplar.

Materials and methods

Plant material and salinity stress treatment

Two P. alba provenances were used for this study. They were collected from natural populations at extreme latitudes of Italy: (i) Bormida river valley from the north of Italy (lat. 44 °21′N, long. 8 °17′E, altitude 400 m above sea level) and (ii) Sinni river valley from the south of Italy (lat. 40 °11′N, long. 16 °42′E, alti-

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tude 20 m above sea level). The original sites differ in ecological conditions and climate parameters such as temperature, rainfall and duration of the summer drought period (Beritognolo et al. 2007). Based on common garden evaluation, genotype 6K3 from Bormida provenance and genotype 14P11 from Sinni prov-enance were further selected. These genotypes were evaluated as genetically divergent by random amplification of polymorphic DNA markers (Sabatti et al. 2001) and also showed a different response to soil salinity, with 14P11 being more salt-tolerant than 6K3 (Beritognolo et al. 2007, Abbruzzese et al. 2009).

For the salt stress experiment, 20-cm-long woody cuttings were prepared during the winter and stored at 0 °C. In spring, the cuttings were individually planted in pots filled with 12 kg of sand (dry weight) for rooting. The average field capacity of the pots was 2.2 l, as determined experimentally. Two-month-old rooted cuttings were used for the experiments. The plants were grown in a greenhouse before and during the experi-ments. Salinity treatment was applied between days of the year 187 and 194. During the experiment, the average temperature was 26.7 °C, and the average minimum and maximum tem-perature was 16.1 and 36.7 °C, respectively. The control plants were watered with half-strength Hoagland’s solution, whereas the salt-stressed plants were watered with 200 mM NaCl solved in half-strength Hoagland’s solution. At the beginning of the experiment, the salinity of pots was rapidly increased by watering five times with 0.5 l of irrigation solution during 2 h, which ensured complete renewal of the pot solution. In the fol-lowing days, each pot was watered twice a day (morning and afternoon) with 0.5 l of irrigation solution. The salinity level was monitored by daily measurement of electrical conductivity (E.Ce.) of the pot percolation solution collected after each irriga-tion. During the time range of the experiment, the E.Ce. values confirmed a salinity level very close to the target concentration of irrigation solutions of control (salt-free 0.5× Hoagland) and salt-treated plants (200 mM NaCl, 0.5× Hoagland). Three destructive samplings (six plants per genotype per treatment) were carried out 3 h, 1 day and 3 days after salt stress initiation. Early mature leaves at positions 7 and 8 from the apex were collected and stored at −80 °C until analysis.

Leaf gas exchange and leaf fluorescence

The physiology of control and salt-stressed plants was studied in vivo using a portable gas-exchange/chlorophyll fluorescence system (Li-Cor 6400-40, Lincoln, NE, USA). Measurements were carried out between 10 am and 2 pm of days 2 and 6 after beginning the salt treatment, on the first fully expanded leaf (i.e., the leaf that had attained its largest size). Measurements were run under controlled conditions, maintaining the leaf in the 2 cm2 gas-exchange cuvette at 40% relative humidity, 30 °C leaf tem-perature, 1000 µmol photons m−2 s−1 incident light intensity and 400 ppm CO2, except during the CO2 response curves. The following parameters were measured: photosynthesis (A), dark

respiration (Rn), intercellular CO2 concentration (Ci), transpira-tion (Tr) and stomatal conductance (gs). The response of pho-tosynthesis to intercellular CO2 (A/Ci curves) was carried out by manipulating the external CO2 concentration between 0 and 1000 ppm with the mixer of the Li-Cor 6400-40 equipment. A/Ci curves were repeated on four leaves of four different plants. The ratio of variable to maximal fluorescence (Fv/Fm) was mea-sured on six replicates per treatment and per genotype after a 30 min adaptation to darkness to yield the maximal efficiency of photosystem II (PSII).

Leaf element composition

Leaf samples were oven dried for 1 day at 65 °C. Sodium (Na+) and potassium (K+) were extracted by adding a small amount of leaf dry mass (50–150 mg) to a 10 ml mixture of HNO3, HClO4 and bi-distilled water (1:5:2.5). The solution was kept at 100 °C overnight, diluted with 100 mM HCl to a final volume of 25 ml and filtered by means of a glass filter before being analyzed by atomic emission spectrometry (I.C.P. Plasma 40, Perkin-Elmer, Monza, MI, Italy). Final concentra-tions were normalized by the weight of the dry leaf sample employed for each extraction. All the extractions were repli-cated twice for each leaf sample.

Analysis of variance and post-hoc tests

Quantitative data of leaf element composition, leaf gas exchange and leaf fluorescence were analyzed by two-way analysis of variance (ANOVA) according to the general linear model, which included genotype and treatment as fixed factors with interac-tion. Post-hoc comparison was performed by Tukey’s honestly significant difference (HSD) test. Time was not included in the model. Therefore, ANOVA and post-hoc tests were performed separately for each time point. Differences were accepted as statistically significant when P < 0.05. Analysis of variance and post-hoc tests were computed with R software (V 2.11.1) (R Development Core Team 2010).

cDNA microarrays

The cDNA microarrays used in this study contained 7342 unique P. euphratica expressed sequence tags (ESTs), which represented 6340 different genes. The genes contained in the array represent a relatively small fraction of the ~45,000 puta-tive genes found in the poplar genome (http://www.phyto-zome.net/poplar). However, these ESTs were selected from P. euphratica cDNA libraries and subtracted cDNA libraries as genes responsive to multiple abiotic stress treatments, includ-ing salt, drought, ozone, cold, freezing and flooding (Brosché et al. 2005); thus the array represents an appropriate tool to investigate the transcriptome response to salinity. The EST arrays were spotted in triplicate on UltraGAPS (Corning Incorporated, Acton, MA, USA) slides at the Turku Finnish microarray center (Turku, Finland).

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RNA extraction, cDNA labeling and array hybridization

Total RNA was extracted using a protocol published by Chang et al. (1993). The extracted RNA was quantified spectrophoto-metrically and stored at −80 °C until analysis. For each sample, 20–24 µg of total RNA was used for cDNA synthesis and label-ing using the Superscript™ Plus Indirect cDNA Labeling System (Invitrogen, San Giuliano Milanese, MI, Italy). Prior to array hybridization, the microarray slides were pre-hybridized in 50 ml of 5× sodium chloride and sodium citrate solution (SSC), 0.1% sodium dodecyl sulfate (SDS) and 1% bovine serum albu-min at 42 °C for 2–5 h. The slides were then washed for 30 s in MilliQ water and 30 s in 95% ethanol. Each labeled sample was re-suspended in 25 µl of 50% formamide, 5× SSC, 0.1% SDS, 0.1 mg ml−1 herring sperm DNA and 5× Denhardt’s solution. The two labelled cDNA samples were mixed, denatured at 96 °C for 2 min and loaded onto the array surface, which was covered with a cover slip and enclosed in a sealed hybridization chamber (model HybChamber™ GeneMachines, San Carlos, CA, USA). The hybridization was carried out at 42 °C overnight. After hybridization the slides were washed in serial baths at room temperature: 2× SSC, 0.1% SDS for 3 min, 1× SSC for 2 min, 0.2× SSC for 1 min and 0.05× SSC for 10 s. The slides were centrifuged to remove any liquid and scanned with a ScanArray Lite instrument (Perkin-Elmer, Monza, MI, Italy). The scanning settings (laser power and photomultiplier tube gain) were adjusted for each slide and each dye channel in order to minimize the number of saturated spots.

The first microarray experiment was conducted according to an RNA pooling design. At each sampling time, 4 µg of total RNA from each of six biological replicates was mixed to obtain a pool (24 µg) that was labeled and hybridized on the array. The hybridizations ‘stress’ versus ‘control’ were carried out for each genotype separately. Three pairs of stress-control pooled samples per genotype were hybridized, which corresponded to 3 h, 1 day, and 3 days of treatment. Secondly, a microarray experiment was conducted on the samples collected at 3 days according to a balanced block design. For each genotype, six labeled samples (20 µg RNA) from biological replicates of stressed plants were individually hybridized against six labeled samples from control plant replicates. Pairs of stress-control samples were randomly assorted and hybridized together. A dye-swap technical replicate was performed for each hybrid-ization in order to compensate for dye-related bias.

Microarray data analysis

The array images were processed with SpotFinder software v. 3.0 (Saeed et al. 2003) to extract the spot signal and local background. In addition to automatic flagging, based on spot quality control, a flagging of bad spots and physical artifacts was manually performed by visual inspection. For each slide, flagged spots and spots with a signal to background ratio below 1.5 were filtered out. Systematic bias in the data was removed

by applying dye-swap normalization (Fang et al. 2003), which makes use of the reverse labelling in the two microarray repli-cates, and the locally weighted scatterplot smoothing (LOWESS) normalization (Yang et al. 2001). The subtraction of the local spot background signal from the foreground signal was done according to the method developed by Scharpf et al. (2007). The same pipeline was used to analyze the first and the second group of experiments, but after the normalization, the datasets of each genotype from the second group of experiments were set together, in order to obtain a higher significance. To estab-lish the significance of observed regulation for each gene, we used Student’s t-test and the P values were corrected for mul-tiple comparisons (Benjamini and Hochberg 1995, Reiner et al. 2003). A χ2 non-parametric test was applied to compare the number of stress-increased ESTs versus the number of stress-decreased ESTs, with a significance level threshold P < 0.05. All the analyses were performed using in-house written R codes (R Development Core Team 2010). Finally, genes with at least a twofold change were considered as salinity regulated.

The functional annotation of array ESTs was carried out with Blast2GO software (Conesa et al. 2005) according to plant-specific Gene Ontology (GO) terms and using the default set-tings. The first step was an automated BlastX search against the NCBI sequence database. A set of 6129 ESTs generated significant Blast results and were submitted to the GO term mapping on the Blast2GO server database. In all, 5234 ESTs were mapped to GO terms and 4465 ESTs received a functional annotation. Finally, the GO terms were converted into a plant-specific GO slim vocabulary to obtain a more organism-specific annotation. Analysis of GO term enrichment between gene sets was performed using Fisher’s exact test implemented in Blast2GO, in order to detect GO terms significantly more fre-quent in a test gene set relative to a reference gene set.

Validation of microarray results with quantitative real-time reverse transcriptase–polymerase chain reaction

Transcripts corresponding to specific genes were quantified by two-step real-time reverse transcriptase–polymerase chain reaction (RT-PCR). The gene-specific primers for PCR were designed on the conserved sequence region using Primer3 soft-ware (Rozen and Skaletsky 2000). The selected primers were analyzed for thermodynamic stability and secondary structures using Vector NTI software (Invitrogen, San Giuliano Milanese, MI, Italy). The specificity of primer pairs was tested by BlastN (Altschul et al. 1997) analysis and in silico PCR against NCBI non-redundant sequence databases and the P. trichocarpa genome sequence (http://www.phytozome.net). The list of prim-ers is provided in Supplementary Material (SM) 1 available as Supplementary Data at Tree Physiology Online. β-Actin was also analyzed as a housekeeping gene (Brunner et al. 2004) and used as reference for normalization and calculation of relative gene expression.

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Total RNA (5 µg) was treated with DNase I and reverse tran-scribed with M-MLV reverse transcriptase enzyme (Invitrogen, San Giuliano Milanese, MI, Italy) following the manufacturer’s instructions. Aliquots of cDNA equivalent to 40 ng of total RNA were used as template for real-time PCR. Reactions were per-formed in triplicate volumes of 15 µl using iQ SYBR Green Supermix reagents and the iQ5 thermal cycler (Bio-Rad, Segrate, MI, Italy). Primer concentration was between 0.3 and 0.5 µM. The amplification thermal program was as follows: one cycle of 3 min at 95 °C for initial denaturation and Taq activation, and 40 cycles of 10 s at 95 °C and 30 s at 56 °C (real-time fluores-cence detection). After amplification, a melting curve cycle was performed with starting temperature at 56 °C, 0.5 °C increment steps, and end temperature at 90 °C. The absence of non-spe-cific PCR products (artifacts and primer dimers) was checked by analysis of the melting curve. When non-specific products were detected, the annealing temperature and primer concentration were adjusted to optimize the specificity of amplification.

Threshold cycle (Ct) was calculated from baseline-sub-tracted RFU data using iQ5 software (Bio-Rad, Segrate, MI, Italy). For each sample the Ct value was averaged over the triplicate reactions. Technical replicates were excluded from data analysis when the Ct range exceeded 0.5 cycles. Polymerase chain reaction efficiency of each amplicon was cal-culated from single-well raw fluorescence data using LinReg software (Ramakers et al. 2003). Polymerase chain reaction efficiency was then averaged over the whole set of samples. The Ct data and PCR efficiency were analyzed with REST soft-ware (V 2.0.7) (Pfaffl et al. 2002) to calculate the statistical significance of the stress/ control ratio.

Results

Leaf mineral content

In leaves of control plants, Na+ concentration was in the range 0.02–0.05 mmol g−1 of dry weight (Figure 1). The Na+ concen-tration in salt-treated plants was several-fold higher than in control plants and the difference was always significant. Na+ concentration built up with increasing exposure to soil salinity and, at 7 days, it reached 0.8 mmol g−1 (~20,000 ppm). The effect of salinity treatment on leaf Na+ concentration was always significant, and the interaction treatment by genotype was significant only at 7 days (ANOVA, SM 2). The leaf Na+ concentration of the two genotypes was very similar in both control and stressed plants. Potassium concentration in leaves did not show significant changes by genotype or treatment effect (SM 2 and SM 3). All values of leaf K+ were in the range 0.2–0.5 mmol g−1 of dry weight.

Leaf gas exchange and chlorophyll fluorescence

The photosynthesis of control plants was relatively low when compared to what is usually observed in poplar leaves in the

field, but very close to that of rooted cuttings in similar growth conditions (Sixto et al. 2005) (Figure 2a). After 2 days of exposure to salinity, we observed a clear and significant reduc-tion of photosynthesis in leaves of both genotypes relative to control plants. At 6 days of the experiment, the reduction of A in salt-treated plants was highly significant and more marked than at 2 days. Moreover, A was significantly lower in salt-treated leaves of the sensitive genotype 6K3 than in leaves of tolerant genotype 14P11. The interaction between treatment and genotype was significant at both time points (SM 2).

Dark respiration was significantly reduced by salinity treat-ment (Figure 2b). At 2 days of the experiment, the respiration in control plants of 14P11 was higher than in salt-treated plants, although the difference was not statistically significant. After 6 days, control plants had significantly greater respiration than salt-treated plants and the interaction between treatment and genotype was significant.

Stomatal conductivity was markedly and significantly reduced by salt treatment (Figure 2c) and treatment and genotype effect were both significant, with 14P11 always showing higher gs than 6K3. The Ci was scarcely affected by salinity treatment (Figure 2d). The effect of salinity treatment was significant only at 2 days, and the difference between salt-treated plants and control plants was significant only in 6K3. The response of photosynthe-sis to varying intercellular CO2 pressure (A/Ci) showed that car-boxylation efficiency was reduced by salinity in both genotypes (Figure 3). The effect was more marked after 6 days than after

Transcriptome response to salinity in Populus alba 1339

Figure 1. Content of Na+ in leaves of two P. alba genotypes (14P11 and 6K3) grown in control conditions (‘c’, solid bars) and at 200 mM NaCl (‘s’, dashed bars). Leaves were sampled 1, 3 and 7 days after the initiation of salinity treatment. Each value is the average of six repli-cated plants and error bars represent standard error. Post-hoc test (Tukey HSD) was performed within samples collected at each time point and different letters indicate values that are significantly different (P < 0.05).

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2 days of treatment. The CO2 compensation point also increased in salt-treated plants after 6 days of treatment.

Chlorophyll a fluorescence was measured to assess the damage to PSII and the overall photochemistry of photosyn-thesis (SM 2 and Figure 4). After 2 days of treatment the effect of salinity was not significant, whereas after 6 days Fv/Fm was markedly and significantly reduced in salinity-treated plants, with no significant differences between genotypes.

Microarray results from pooled RNA design

The array used in this study contains 7342 ESTs representing 6340 genes, which are a fraction of the poplar genome; how-ever, it has previously proved successful in identifying stress-related genes (Brosché et al. 2005; Brinker et al. 2010). The first step of microarray experiments used an RNA pooling design. This analysis revealed that, in both genotypes, the number of salinity-regulated ESTs increased from 3 h to 3 days of treat-ment (Figure 5). At each time point the number of ESTs with decreased expression was larger than that of ESTs with increased expression. The 3-day time point had the highest

number of regulated genes and was considered as the most informative sampling. For this reason a detailed analysis was conducted on individual plants collected at this time according to a balanced block design, which allowed a statistical analysis of transcriptome changes induced by salt stress.

Microarray results from balanced block design

The results of balanced block microarray experiments on plants sampled at 3 days revealed a similar number of ESTs detected (i.e., where a significant signal was obtained from the array) in the two genotypes: 5563 (75.4% of total array) and 5617 (76.2% of the array), of which 5070 and 5154 were retained as the dataset for statistical analysis, respectively in 14P11 and 6K3. The datasets of the two genotypes largely overlapped (4836 shared ESTs). In all further analyses, the ‘reference’ dataset of each genotype is composed of all ESTs from the array with a strong enough signal to be detected.

Lists of all ESTs significantly regulated by salinity are pro-vided in SM 4. The overlap of the salinity-regulated ESTs from the two genotypes is illustrated by Venn diagrams in Figure 6. The number of significant ESTs was very similar in the two

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Figure 2. Leaf gas exchange in two P. alba genotypes (14P11 and 6K3) grown in control conditions (‘c’, solid bars) and at 200 mM NaCl (‘s’, dashed bars). Measurements were taken at 2 and 6 days after initiation of salinity treatment. Each value is the average of six replicated plants and error bars represent standard error. Post-hoc test (Tukey HSD) was performed within samples collected at each time point and different letters indicate values that are significantly different (P < 0.05). (a) Net photosynthetic assimilation (A). (b) Respiration in the dark (Rn). (c) Stomatal conductance (gs). (d) Intercellular partial pressure of CO2 in the mesophyll (Ci).

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genotypes: 1066 in 14P11 (549 increased and 517 reduced expression) and 1019 in 6K3 (413 increased and 606 reduced expression). In 6K3, the number of ESTs with decreased expression was significantly higher (χ2 test) than that of ESTs with increased expression, whereas the difference was not sig-nificant in 14P11. When a twofold change between stress and control was applied as a threshold filter, about 20% of the significant ESTs were retained as differentially expressed, 196 ESTs in 14P11 and 222 ESTs in 6K3, with no significant differ-ence between genotypes. In both genotypes the number of ESTs with decreased expression was significantly larger than that of ESTs with increased expression. Most genes with decreased expression were common to the two genotypes, whereas many genes with increased expression were specific to each genotype. After the global analysis above, the genes with at least twofold expression change in salt-stressed leaves relative to control were studied in more detail.

Shared genes

Of the 149 ESTs that were salt regulated in both genotypes, 130 ESTs had decreased expression. Of this set, 114 ESTs were annotated and corresponded to 71 different genes. In

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Figure 3. Response of net photosynthetic assimilation to increasing CO2 partial pressure (A/Ci curve) in leaves of two P. alba genotypes (14P11 and 6K3) grown in control conditions (0.5× Hoagland) and at 200 mM NaCl. Each curve corresponds to an individual plant. Measurements were taken at 2 and 6 days after initiation of salinity treatment. (a, b) Results after 2 days. (c, d) Results after 6 days.

Figure 4. Variable to maximum chlorophyll fluorescence ratio (Fv/Fm) in leaves of two P. alba genotypes (14P11 and 6K3) grown in control conditions and at 200 mM NaCl. Measurements were taken at 2 and 6 days after initiation of salinity treatment. Each value is the average of six replicated plants and error bars represent standard error. Post-hoc test (Tukey HSD) was performed within samples collected at each time point and different letters indicate values that are significantly dif-ferent (P < 0.05).

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order to simplify the presentation of results, redundant ESTs were averaged if they corresponded to the same protein (i.e., proteins of PSI and II, enzyme subunits) and the final gene list

was ordered according to GO annotation of the biological pro-cess (Table 1). A relatively small number of ESTs (19) had increased expression in both genotypes. They corresponded to 13 different genes, mainly involved in response to stress and transport. The shared genes with decreased expression were mainly involved in photo synthesis, response to stress, carbohydrate metabolic processes and generation of precursor metabolites and energy.

Genotype-specific genes

Thirty-three ESTs were specifically increased by salinity only in the salt-tolerant 14P11 (Table 2) and corresponded to 21 dif-ferent genes, which were mainly involved in response to stress, transport and biosynthetic process. The 14 transcripts with decreased expression in 14P11 corresponded to eight differ-ent genes involved in various biological processes, mainly related to metabolism. In contrast to 14P11, the ESTs specifi-cally salt-regulated in the salt-sensitive 6K3 included more genes with decreased than increased transcript level (Table 3). Only 15 ESTs had increased expression specifically in 6K3 and corresponded to 11 different genes, mainly involved in response to stress and the nucleoside, nucleotide and nucleic acid metabolic processes. Fifty-three ESTs had decreased expression in 6K3 and corresponded to 43 different genes. The main biological processes related to these genes were electron transport, secondary metabolic processes (in particu-lar flavonoid biosynthesis), photosynthesis and metabolic processes.

Functional annotation of salinity-regulated ESTs

A concise description of the functional annotation was obtained by calculating the relative proportion of each GO term at hier-archy level 2, which is the highest node of each GO category. This annotation summarizes the functional information and facilitates the comparison among gene sets and species (Figure 7 and SM 5). The GO term partitioning of genes dif-ferentially expressed in response to salinity showed a deviation from the array annotation. The partitioning of GO terms was very similar in ESTs with decreased expression of both geno-types. In contrast, the annotation profile of ESTs with increased expression was markedly different between the two genotypes in the three GO sections: biological process, molecular function and cellular component.

GO term enrichment analysis

Fisher’s exact test was performed to identify GO terms that were significantly more represented among the salt-regulated genes compared to the reference set (SM 6). When consider-ing genes with increased expression, the enrichment analysis revealed differences between the genotypes (SM 6a). Enriched GO terms were mainly in the biological process section, with common enriched terms related to metabolic processes,

1342 Beritognolo et al.

Figure 5. Number of ESTs with expression significantly (t-test, P < 0.05) increased or decreased by salinity in leaves of two P. alba genotypes (14P11 and 6K3) after 3 h , 1 day and 3 days of exposure to 200 mM NaCl, relative to control conditions. Microarray analyses carried out on pooled RNA samples from six plants per genotype and per treatment.

Figure 6. Venn diagrams comparing the ESTs with expression signifi-cantly increased (left plots) or decreased (right plots) by salinity stress in leaves of two P. alba genotypes (14P11 and 6K3) after 3 days of exposure to 200 mM NaCl. The top row represents all signifi-cant ESTs (t-test, P < 0.01). The bottom row represents significant ESTs with at least twofold change relative to the control. Up: salinity-increased expression. Down: salinity-decreased expression.

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Table 1. Microarray results of selected genes with expression significantly increased (a) or decreased (b) by salinity stress in both P. alba geno-types with at least twofold change (FC) relative to control.

EST accession

Annotation/description (BlastX hit) Biological process (GO term) 14P11 FC 6K3 FC

Common salinity-increased ESTsAJ778201 glutathione reductase Response to stress 3.558 2.286AJ779375 rubber elongation factor/stress related protein Response to stress 2.513 2.102AJ778500 homeobox-leucine zipper protein Response to stress 2.033 2.064AJ780291 pheophorbide a oxygenase, chloroplast precursor

(lethal leaf-spot 1 homolog lls1)Response to stress 4.016 2.471

AJ780749 plastid-lipid-associated protein Response to stress 2.972 2.6422 ESTs progesterone 5-beta-reductase Metabolic process 2.450 2.2032 ESTs ATSIP2 (A. thaliana seed imbibition 2) hydrolyzing

O-glycosyl compoundsCarbohydrate metabolic process 2.638 4.528

2 ESTs bark storage protein Nucleobase, nucleoside, nucleotide and nucleic acid metabolic process

3.121 7.053

AJ771347 ABC transporter Transport 2.548 2.146AJ770033 ferritin light chain Transport 3.125 3.8363 ESTs zinc finger (C3HC4-type ring finger) family protein Unknown 4.446 2.751AJ779884 geko1-like ethanol tolerance protein Unknown 2.894 2.121AJ778618 glycosyltransferase UGT88A8 Unknown 3.304 2.557

Common salinity-decreased ESTs2 ESTs carbonic anhydrase Metabolic process −5.358 −7.290AJ769169 diphosphonucleotide phosphatase Metabolic process −2.156 −2.545AJ772833 mRNA binding protein precursor Metabolic process −2.229 −2.030AJ768869 magnesium chelatase subunit Biosynthetic process −2.105 −2.905AJ780833 lipase family protein Catabolic process −2.587 −2.844AJ769206 glutamyl-tRNA reductase Amino acid and derivative metabolic process −2.003 −2.038AJ769310 hydroxypyruvate reductase Amino acid and derivative metabolic process −2.250 −3.230AJ768072 p-protein precursor Amino acid and derivative metabolic process −2.710 −3.3684 ESTs glycine decarboxylase cleavage complex Amino acid and derivative metabolic process −3.075 −4.076AJ770034 lon protease Protein metabolic process −3.060 −3.605AJ770522 serine carboxypeptidase S28 family protein Protein metabolic process −2.171 −2.129AJ770832 fructose-bisphosphate aldolase class-I Carbohydrate metabolic process −3.248 −3.4832 ESTs glyceraldehyde-3-phosphate dehydrogenase Carbohydrate metabolic process −2.248 −2.493AJ780349 phosphoglycerate kinase Carbohydrate metabolic process −2.648 −3.412AJ768493 malate dehydrogenase Carbohydrate metabolic process −3.057 −3.0744 ESTs plastidic aldolase Carbohydrate metabolic process −4.187 −4.8852 ESTs starch synthase Carbohydrate metabolic process −3.787 −2.7472 ESTs transketolase 1 Carbohydrate metabolic process −2.165 −2.099AJ767913 chalcone synthase Secondary metabolic process −2.282 −3.3952 ESTs ATP synthase subunit Generation of precursor metabolites and energy −2.549 −3.496AJ768537 H+-transporting ATP synthase chain 9-like prot. Generation of precursor metabolites and energy −2.067 −2.651AJ767424 NADH dehydrogenase subunit k Generation of precursor metabolites and energy −2.076 −2.428AJ769268 phosphoglycerate kinase Generation of precursor metabolites and energy −3.135 −4.418AJ767912 carbohydrate binding catalytic Generation of precursor metabolites and energy −2.976 −3.012AJ770175 phosphatidylinositol phosphatidylcholine transfer protein Transport −2.735 −2.477AJ772852 proline-rich protein Transport −5.268 −7.696AJ776715 (ex. protein) glycolipid transfer protein-like Transport −3.669 −4.773AJ768030 (ex. chloroplast precursor) plastocyanin precursor Electron transport −2.599 −2.516AJ778848 chloroplast precursor Electron transport −2.298 −2.4314 ESTs ferredoxin i precursor Electron transport −3.61611 ESTs chlorophyll a b-binding protein Photosynthesis −3.510 −4.47210 ESTs PSI protein Photosynthesis −2.276 −2.7947 ESTs PSII protein Photosynthesis −2.626 −3.3817 ESTs rubisco subunits Photosynthesis −3.972 −4.4568 ESTs rubisco activase Photosynthesis −3.524 −5.048AJ778941 ultraviolet-B-repressible protein Photosynthesis −3.191 −4.455AJ767958 glyceraldehyde-3-phosphate dehydrogenase A subunit Photosynthesis −2.406 −3.223

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Table 1. Continued

EST accession

Annotation/description (BlastX hit) Biological process (GO term) 14P11 FC 6K3 FC

AJ774601 ultraviolet-B-repressible protein Photosynthesis −2.346 −3.3803 ESTs galactinol synthase Response abiotic stimulus −7.919 −5.530AJ778904 metallothionein-like protein Response to stimulus −2.161 −2.268AJ780605 benzoquinone reductase-like protein Response to stress −2.422 −3.135AJ768555 BURP domain-containing protein/RD22-like protein Response to stress −2.887 −2.8472 ESTs peroxidase PRXR1 Response to stress −3.896 −3.625AJ773781 quinone oxidoreductase Response to stress −2.114 −2.290AJ779407 two-component response regulator Response to stress −2.347 −2.639AJ774487 germin-like protein Signal transduction −2.427 −4.125AJ773957 RAB7 family small GTPase Signal transduction −2.702 −2.491AJ776024 40S ribosomal protein S20 Translation −3.694 −4.007AJ767823 ribosomal protein l3 Translation −2.525 −2.249AJ776088 hyoscyamine 6 beta-hydroxylase Unknown −2.515 −2.444AJ769598 photoassimilate-responsive protein par-like protein Unknown −2.820 −2.961AJ774761 pumilio Mpt5 family RNA-binding protein Unknown −3.653 −4.008AJ780488 senescence-inducible chloroplast stay-green protein Unknown −2.910 −4.760

Table 2. Microarray results of selected genes with expression significantly increased or decreased by salinity only in genotype P. alba 14P11, with at least twofold change (FC) relative to control.

EST accession Annotation/description (BlastX hit) Biological process (GO term) 14P11 FC 6K3 FC

Salinity-increased4 ESTs heat shock protein 70 Response to stress 2.137 1.694AJ780435 aldehyde dehydrogenase 7B4 Response to stress 2.555 2.491 nsAJ778125 basic endochitinase/PR protein Response to stress 2.523 1.674AJ779386 osmotin-like protein Response to stress 4.773 2.358 ns2 ESTs putative early light induced protein Response to stress 2.252 1.087 nsAJ768562 BEL-1 like homeodomain transcription factor Transcription 2.318 1.663AJ770335 formate dehydrogenase Metabolic process 6.076 10.197 nsAJ780649 sucrose synthase Carbohydrate metabolic process 3.434 4.489 nsAJ777261 asparagine synthetase Biosynthetic process 4.036 3.281 nsAJ779916 trehalose-6-phosphate synthase Biosynthetic process 2.639 2.255 nsAJ777708 peptide methionine sulfoxide reductase Protein modification process 2.138 1.8332 ESTs plasma membrane intrinsic protein Transport 2.344 0.239 nsAJ769587 P-type H+-ATPase Transport 2.033 1.267 nsAJ778450 vacuolar H+-ATPase catalytic subunit A Transport 2.353 1.550AJ777018 SEC14 cytosolic factor family protein/phosphoglyceride

transfer family proteinTransport 2.130 1.091 ns

AJ779430 expansin-like protein Cell growth 2.414 1.540 nsAJ776998 flavonol sulfotransferase Unknown 2.422 1.006 ns2 ESTs Kunitz trypsin inhibitor ti3 Unknown 2.860 1.669 ns2 ESTs polyphenol oxidase Unknown 9.350 5.152 ns

Salinity-decreasedAJ772160 BRH1 ring finger protein Response to stimulus −2.025 −1.158 nsAJ773352 AP2 ERF domain-containing transcription factor Signal transduction −3.847 −1.127 nsAJ769015 ADP-glucose pyrophosphorylase large subunit Carbohydrate metabolic process −2.010 −1.931AJ773904 ccr4-associated factor Nucleobase, nucleoside, nucleotide and

nucleic acid metabolic process−2.708 −1.433 ns

2 ESTs EF-hand Ca2+ -binding protein ccd1 Protein metabolic process −2.475 −1.146 nsAJ773252 glutamine synthetase Amino acid and derivative metabolic

process−2.052 −1.904

AJ768055 psan (PSI reaction center subunit PSI-n) calmodulin binding

Photosynthesis −2.756 −2.990 ns

AJ774495 neoxanthin cleavage enzyme-like protein Unknown −2.095 −1.571

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Table 3. Microarray results of selected genes with expression significantly increased or decreased by salinity only in genotype P. alba 6K3, with at least twofold change (FC) relative to control.

EST accession Annotation/description (BlastX hit) Biological process (GO term) 14P11 FC 6K3 FC

Salinity-increasedAJ779923 late embryogenesis-abundant protein (LEA) Response to stress 1.567 ns 4.961AJ780896 fiber protein, universal stress protein (USP) Response to stress 1.446 ns 2.455AJ772947 NAC domain protein Transcription 1.512 2.198AJ775563 60S ribosomal protein L10 Translation 1.500 2.075AJ770731 benzoquinone reductase Response to endogenous stimulus 1.722 2.061AJ771345 seed imbibition protein/alkaline alpha

galactosidaseCarbohydrate metabolic process 1.743 2.172

AJ772697 bark storage protein Nucleobase, nucleoside, nucleotide and nucleic acid metabolic process

4.036 ns 8.882

AJ774050 methylthioadenosine/S-adenosyl homocysteine nucleosidase (MTA)

Nucleobase, nucleoside, nucleotide and nucleic acid metabolic process

1.482 2.053

AJ776278 peptidylprolyl isomerase Unknown 1.614 2.051AJ780103 exostosin-like glycosyltransferase Unknown 1.155 ns 2.858

Salinity-decreasedAJ773215 class I chitinase Response to stress −1.471 −2.305AJ768200 S-adenosyl-L-homocysteine hydrolase Response to stress −1.392 ns −2.232AJ767610 LrgB-like family protein Signal transduction −1.507 −2.240AJ776717 anthocyanidin reductase Metabolic process −1.307 −2.363AJ779238 leucoanthocyanidin reductase Metabolic process −1.994 −2.819AJ775026 glucosyltransferase homolog Metabolic process −1.587 −2.3222 ESTs xyloglucan endotransglycosylase Carbohydrate metabolic process −0.359 ns −2.3642 ESTs 3-deoxy-D-arabino-heptulosonate 7-phosphate

synthaseAmino acid and derivative metabolic process −1.831 −2.704

AJ770499 glutamine synthetase Amino acid and derivative metabolic process −1.976 −2.300AJ774686 ATP synthase gamma-subunit Generation of precursor metabolites and energy −1.944 −2.0123 ESTs phenylalanine ammonia-lyase Secondary metabolic process −1.592 −3.174AJ767478 flavanone 3-hydroxylase Secondary metabolic process −1.337 −3.710AJ777465 dihydroflavonol 4-reductase Secondary metabolic process −1.084 ns −2.016AJ768390 phytoene synthase Secondary metabolic process −1.704 −2.073AJ767357 nucleotide pyrophosphatase-like protein Catabolic process −1.938 −2.019AJ772360 thiazole biosynthetic enzyme Biosynthetic process −3.164 ns −2.7455 ESTs PSI protein Photosynthesis −1.788 −2.2283 ESTs PSII protein Photosynthesis −1.647 −2.496AJ773283 Sep2 (stress enhanced protein 2)/chlorophyll

binding proteinPhotosynthesis −1.922 −2.268

AJ769407 dicyanin blue copper protein Electron transport −1.952 −2.2592 ESTs elicitor-inducible cytochrome p450 Electron transport −1.326 −2.3192 ESTs nitrate reductase Electron transport −1.442 −2.214AJ768182 glycolate oxidase Electron transport −1.933 −2.615AJ775080 cytochrome b6 f complex subunit iv Electron transport −1.989 −2.357AJ768812 cytochrome b6-f complex rieske iron-sulphur

proteinElectron transport −1.645 −2.153

AJ778650 ferredoxin-NADP reductase Electron transport −1.905 −2.942AJ773383 extensin-like protein/proline rich protein Cellulose and pectin-containing cell wall

organization and biogenesis−1.233 ns −2.567

AJ780807 trafficking protein particle complex 2-like Transport −1.706 −2.227AJ773340 protease inhibitor/seed storage/lipid transfer

protein (LTP)Transport −1.828 −2.135

AJ767812 atbpm3 protein binding Unknown −1.788 −2.177AJ769365 NADH-plastoquinone oxidoreductase subunit j Unknown −1.275 ns −2.004AJ778373 sah7 pollen protein Unknown −1.270 ns −2.076AJ768378 small conjugating protein ligase Unknown −1.618 −2.085AJ767661 enh1 (enhancer of sos3–1) Unknown −1.915 −2.157

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translation and cellular biosynthesis. Enriched GO terms spe-cific to 14P11 were related to response to exogenous stimuli, catabolism, cell death and development, whereas 6K3-specific terms were related to metabolic processes, biosynthesis and biogenesis. For the cellular component, both genotypes were enriched in terms related to non-membrane-bound organelles and macromolecular complexes. Enriched terms specific to 14P11

were vacuole and plasma membrane, whereas 6K3-specific terms were related to cytoplasm and intracellular compart-ments. In molecular function, both genotypes were enriched in terms related to nucleic acid binding and translation. The molecular functions specific to 14P11 were related to protein hydrolase and catalytic activity, whereas the terms specific to 6K3 were RNA binding and structural molecule activity.

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Figure 7. Partitioning of GO functional categories in ESTs increased or decreased by salinity in leaves of two P. alba genotypes (14P11 and 6K3) after 3 days of exposure to 200 mM NaCl. Relative abundance of GO terms at the highest node (level two) of GO hierarchy. P, genotype 14P11; K, genotype 6K3; det, detected ESTs; inc, increased-expression ESTs; dec, decreased-expression ESTs.

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For genes with decreased expression, the changes in GO terms were very similar in the two genotypes with the same list of enriched GO terms (SM 6b). The shared enriched biological processes include primary and energy metabolism, biosynthe-sis and response to abiotic stimulus. Small differences between genes with decreased expression of the two genotypes were observed only in the biological process. Three GO terms were enriched only in 6K3: amino acid and derivative metabolic pro-cess, secondary metabolic process and catabolic process. The only enriched molecular function was catalytic activity in both genotypes.

Validation of microarray results by quantitative real-time RT-PCR

Twelve genes, representative of different transcription profiles, were selected for validation by quantitative real-time RT-PCR (SM 7). For nine genes, real-time PCR results validated the array results in both genotypes, considering increased or decreased transcript level and statistical significance. The genes EF-hand Ca2+-binding protein ccd1 and glutathione S-transferase were validated in 6K3, whereas in 14P11 the real-time PCR results showed the same change (increased or decreased tran-script level), but the statistical significance was not confirmed. The Peroxidase gene was statistically validated in 14P11, but not in 6K3; however, the trend was consistent in both array and real-time PCR analyses. Overall, the genes with more than two-fold change in the transcript level were more consistently validated than genes with less than twofold change.

Discussion

Effect of salinity on leaf ion balance

The Na+ content in leaves of salt-treated plants significantly increased as early as 1 day after stress initiation and built up with time. These data are consistent with previous observa-tions in P. alba (Sixto et al. 2005, Mao et al. 2008, Imada et al. 2009) and other Populus species (Chen et al. 2001, Brosché et al. 2005, Mao et al. 2010). The leaf Na+ content observed after 7 days of exposure to 200 mM NaCl was close to 0.8 mmol g−1 of dry matter. This value is higher than what was previously observed in the same genotypes after 1 month of exposure to soil salinity (Beritognolo et al. 2007) and is com-parable to the Na+ content of P. euphratica trees naturally growing in saline soils (Brosché et al. 2005).

The ability of plants to survive in saline environments is linked to their capacity to maintain a stable Na+:K+ ratio (Munns and Tester 2008). In this experiment, the Na+:K+ ratio increased in salt-stressed plants above 1:1, as previously observed in P. alba by Sixto et al. (2005). The accumulation of Na+ ions often competes against absorption and uptake of K+, which further increases the Na+:K+ ratio. In P. alba Imada et al. (2009) observed a reduction of leaf K+ after 1 year of exposure to soil

salinity. In our P. alba plants, the leaf K+ content was not signifi-cantly reduced by salt stress, which is consistent with previous studies on P. alba plants exposed to soil salinity for some weeks (Mao et al. 2008, 2010).

Despite strong stomatal closure and low transpiration, salt rapidly accumulated in leaves of the P. alba genotypes. This indicates that Na+ ions are rapidly taken up from the soil and translocated from roots to leaves through the transpiration flux. The exclusion of salt at the root level is a well-established tol-erance mechanism of P. euphratica, a poplar species that toler-ate very high salinity levels (Chen et al. 2001, 2002, 2003, Sun et al. 2010a). Independent experiments with P. alba geno-types of diverse origin showed that this species takes up Na+ from the soil to the shoot, but Na+ exclusion at the root level is efficient at low salinity (50 mM) (Mao et al. 2008, 2010, Imada et al. 2009). Moreover, an intra-specific variation in Na+ uptake within P. alba species has been reported, with some geno-types accumulating less Na+ in leaves (Sixto et al. 2005, Beritognolo et al. 2007). Although not comparable with P. euphratica in terms of tolerance to high salinity, P. alba may be suitable for plantations in saline soils, provided that geno-types also capable of salt exclusion in roots are selected.

Although the accumulation of Na+ in leaves reached similar values in the two genotypes, they differed in terms of the expression of genes related to ion transport across mem-branes. Transcripts homologous to plasma membrane (P-type) proton pumps and tonoplast (V-type) proton pumps specifi-cally increased in salt-stressed plants of the tolerant genotype 14P11. A differential expression of P-type and V-type proton pump genes between these P. alba genotypes was already observed in a previous study (Beritognolo et al. 2007). These proteins hydrolyze ATP to actively pump protons in the apo-plast and in the vacuole, respectively. This proton gradient can energize the Na+/H+ antiporters, which actively transport Na+ ions from the cytosol into the vacuole or in the apoplast (Blumwald 2000, Blumwald et al. 2000, Tester and Davenport 2003, Zhu 2003). These mechanisms are relevant for salinity tolerance in poplar. The salt-tolerant P. euphratica showed a high activity of P-type H+-ATPases in leaves, which was main-tained after long exposure to soil salinity. In contrast, the salt-sensitive P. popularis showed a progressive decline of PM H+-ATPase, which resulted in a higher Na+ content in the cyto-plasm of leaf cells (Ding et al. 2010, Ma et al. 2010). In P. euphratica, it was also observed that the excess of Na+ is excluded in the apoplast and accumulates in the cell wall (Ottow et al. 2005a). Given the interconnection with proton pumps, it is expected that the expression of N+/H+ antiporters would also be affected by salinity. In this experiment, the array ESTs homologous to sodium antiporters were not significantly salinity-regulated (SM 4). However, the increased expression of proton pump genes in 14P11 could be an advantage for active Na+ exclusion. Further microanalysis of cellular

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localization of Na+ could provide relevant information about the effective sites of sodium accumulation.

Effect of salinity on photosynthesis

Photosynthesis of control and salt-stressed plants was very simi-lar to that observed in rooted cuttings in similar growth condi-tions (Sixto et al. 2005, Mao et al. 2008, 2010), thus ruling out a specific impact of growth conditions on salt-stress effect on plant physiology. In previous experiments, the same P. alba gen-otypes used here showed contrasting responses to progressive salinity stress, with the southern one (14P11) being more toler-ant than the northern one (6K3), thanks to physiological and developmental adaptive changes that allowed a better survival and a higher growth (Beritognolo et al. 2007, Abbruzzese et al. 2009). In this experiment, the response to salt stress was quite similar and a strong inhibition of photosynthesis was observed in both genotypes, although the northern genotype was signifi-cantly more inhibited by salinity after 6 days. Since the salinity treatment was applied as a shock stress, plants suddenly faced a high salinity level (200 mM) without a previous acclimation period. These harsh conditions, representative of sea water intrusion in the water table of coastal areas, may have flattened the difference between genetically contrasting genotypes.

Causes for photosynthesis inhibition by soil salinity include stomatal closure (Delfine et al. 1999), reduced mesophyll conductance, ion toxicity and membrane disruption (Di Martino et al. 2003). Stomatal closure may be driven by abscisic acid (ABA) signaling from roots due to the osmotic component of salinity stress. This signaling pathway reacts at the very early stages of stress sensing (Chen et al. 2003, Wang et al. 2007). In P. alba plants, salt stress caused a strong stomata closure already 2 days after stress initiation. However, the Ci was only transiently reduced, after 6 days again being similar in control and salt-stressed leaves. Therefore, whereas stomatal closure might have contributed to reduced photosynthesis 2 days after the treatment, it did not directly limit photosynthesis after 6 days.

The reduction of mesophyll conductance under salt stress could also lead to a chloroplast CO2 concentration (Cc) lower than Ci (Flexas et al. 2004), eventually reducing the CO2 avail-able at rubisco’s sites in a way that is not visible by assessing CO2 concentration in the intercellular spaces. Low mesophyll conductance was observed to limit photosynthesis of salt-stressed Spinacia oleracea L. (Delfine et al. 1999) and Olea europaea L. (Loreto et al. 2003). Indication that this might also limit photosynthesis of salt-stressed P. alba is provided by gene expression results. The carbonic anhydrase gene had markedly decreased expression in both genotypes (Table 1). Carbonic anhydrase catalyzes the interconversion of CO2 and HCO3

− and facilitates the diffusion of CO2 across the chloro-plast membranes (Moroney et al. 2001), thus affecting meso-phyll conductance (Gillon and Yakir 2000). The strong

decrease of carbonic anhydrase transcripts also suggests that mesophyll-diffusive limitations to photosynthesis occurred in both salt-stressed genotypes.

The experimental data suggest that biochemical and photo-chemical limitations reduced photosynthesis, although with a different time course. Salinity can affect photosynthesis by alteration of biochemistry or photochemistry of carbon assimi-lation (Chaves et al. 2009). This may be the case especially when a rapid accumulation of Na+ in leaves occurs, as in our P. alba plants. Sodium ions at concentration >100 mM inhibit the activity of many enzymes involved in photosynthesis (Munns et al. 2006) and an increased Na:K ratio can inhibit enzymes that require K+ as a cofactor (Blumwald 2000, Tester and Davenport 2003). The drop in the linear response of photosynthesis to intercellular CO2 concentration (A/Ci) showed that rubisco activity was inhibited by salinity since the early phases of salinity stress (Delfine et al. 1999). Consistently, genes encoding rubisco subunits, rubisco activase, and plas-tidic aldolase (AldP), which participate in CO2 fixation, had a marked decrease of expression in both genotypes (Table 1). A transient decrease of the transcripts of some AldP isoforms was also observed in Nicotiana spp. in the early phases of salt stress, and it was proposed as a mechanism for down-regulat-ing photosynthesis in response to a limiting stress (Yamada et al. 2000). Given the strong inhibition of photosynthesis, the marked down-regulation of AldP expression in our salt-stressed plants could indeed be related to the down-regulation of the photosynthetic machinery.

The A/Ci responses also show a clear increase of CO2 com-pensation point (the point at which photosynthesis compen-sates for photorespiration and mitochondrial respiration) at 6 days after stress initiation. Thus, the processes emitting CO2 were not as strongly inhibited as photosynthesis. A significant reduction of leaf dark respiration (Rn) was observed in salt-stressed leaves. Thus, we speculate that photorespiration was mainly responsible for the increased compensation point. Since the specificity factor of rubisco for carboxylation and oxygen-ation is believed to be constant, our finding may indicate that the ratio between substrates in the chloroplasts has been affected by salt stress, with substantial reduction of CO2 con-centration, thus confirming the importance of mesophyll diffu-sion resistance in salt-stressed leaves (Delfine et al. 1999, Loreto et al. 2003, Flexas et al. 2004). Photorespiration might also have been enhanced by increasing leaf temperature in stressed leaves. Under the experimental conditions (summer) the strong stomatal closure might have indeed limited heat dis-sipation by transpiration and increased leaf temperature.

The excess of salt can also limit the photochemical reactions by inhibition of electron transport and damage to the ultra-structure of PSII (Murata et al. 2007, Takahashi and Murata 2008, Mehta et al. 2010). The functionality of PSII was moni-tored by the chlorophyll a fluorescence parameter (Fv/Fm),

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which measures PSII quantum yield. This parameter signifi-cantly decreased in stressed plants after 6 days of treatment, indicating structural damage to PSII. Such damage often occurs after long exposure to salinity (Sixto et al. 2006) and indicates photoinhibition, which develops when the reducing power pro-duced by photochemical reactions is greater than its use by the Calvin cycle. Photoinhibition generates ROS, which, if not efficiently detoxified by foliar antioxidants, may further damage photosynthesis by inhibition of PSII repair (Nishiyama et al. 2006, Takahashi and Murata 2008). As ROS are produced under enhanced oxygenation capacity, the observed increase of CO2 compensation point in salt-stressed leaves may be associated with the photochemical damage that occurred with a similar time course.

Functional annotation of stress-regulated genes

The analyses of functional annotation (GO terms) were per-formed on ESTs with significant increase or decrease of expression in salt-stressed plants, without a fold change threshold of salinity/control ratio. However, even small changes of many genes contributing to the same pathway or to the same biological process can lead to significant phenotypic changes. In this regard, we caution that the analysis of func-tional annotation summarizes the effect of complex changes in transcriptomes. The partitioning of the GO term was very simi-lar in ESTs with decreased expression of the two genotypes. In contrast, the functional annotation of ESTs with increased expression discriminated the two genotypes in terms of tran-scriptome response (Figure 7).

The GO terms that were more represented in genes with salinity-decreased expression were almost the same in both genotypes (SM 6b). The biological processes of this gene set were related to carbohydrate metabolism, energy metabolism and photosynthesis, in agreement with the strong inhibition of leaf functionality revealed by the ecophysiology data. No GO terms were over-represented in transcripts specifically decreased in 14P11, and this indicates that they were functionally related to the common effect of salinity. On the contrary, the ESTs with decreased expression in 6K3 were functionally related to amino acid metabolism, secondary metabolism and catabolism.

The comparative analysis of GO term enrichment in ESTs with increased expression revealed interesting commonalities and differences between the genotypes (SM 6a). Some GO terms were enriched in both genotypes. The biological pro-cesses of this group were related to biosynthesis of proteins and macromolecules, and primary metabolism. The cellular components (macromolecule complex and protein-based com-plexes) and the molecular functions (nucleic acid binding and translation) enriched in this EST set addressed mainly protein synthesis.

When considering the ESTs with increased expression in genotype 6K3, most enriched GO terms were functionally

similar to what was observed in both genotypes: biosynthesis, and metabolism of proteins and macromolecules. Only two GO terms, cellular organization and biogenesis (BP) and structural molecule activity (MF), were specifically enriched in 6K3. This suggests that the formation of new cell structures was stimu-lated in this genotype. In contrast to 6K3, the GO terms enriched in 14P11 were distinct from the common enriched terms. The biological processes, stress induced in this geno-type, were related to cell development, cell death, catabolism, secondary metabolism and stress response. These terms sug-gest a rearrangement of metabolism and cell development in leaves. The up-regulation of catabolic processes could be related to the degradation and turnover of damaged cell struc-tures. The induction of cell death and development processes could be required to adjust leaf growth as a short-term adapta-tion to the stress. Catabolic process and secondary metabo-lism were regulated in an opposite manner between these genotypes. Thus, they represent interesting processes for further investigation. The cellular components specifically enriched in 14P11 (plasma membrane and vacuole) were related to the active transport across membranes. This is con-sistent with the increased expression of P-type and V-type pro-ton pumps in this genotype. The molecular functions that characterized 14P11 were related to enzymatic activities such as catalysis and hydrolysis, instead of structural proteins that were specific to the other genotype.

Genes regulated by salinity in both genotypes

The expression of most of the genes significantly affected by salinity decreased in both genotypes. By their functional anno-tation, these genes were mainly attributed to photosynthesis and carbohydrate metabolism (Table 1). This result is consis-tent with the strong inhibition of photosynthesis observed in the two P. alba genotypes. The expression of carbonic anhy-drase, plastidic aldolase, rubisco subunits and rubisco activase was strongly decreased by salinity in both genotypes, and helps to explain the inhibition of photosynthesis, as discussed above.

Some genes with decreased expression are functionally related to stress responses. The transcripts of galactinol syn-thase were strongly decreased in both P. alba genotypes. This enzyme catalyzes the first step in the synthesis of raffinose family oligosaccharides. In contrast, increased galactinol syn-thase expression was observed in P. euphratica under salinity stress, but the increase of compatible osmolytes was not related to osmotic adjustment in this species (Ottow et al. 2005b). Another possible physiological role of galactinol syn-thase is to produce osmoprotectants that stabilize the mem-branes and protect against oxidative stress (Vinocur and Altman 2005). In addition, the expression of a gene encoding a proline-rich protein (EST AJ772852) was strongly decreased by salinity in both P. alba genotypes. A comparative analysis of

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transcriptomes in rice genotypes revealed the differential up-regulation of proline-rich protein in a salt-tolerant genotype relative to a salt-sensitive one. It was proposed that this protein could have a protective role in the stability of the cell wall against physical stress occurring during salinity stress (Roshandel and Flowers 2009). The decreased expression of these genes suggests that in P. alba the major down-regulation of metabolic pathways in leaves impaired some protection mechanisms and likely contributed to damaged photosynthesis.

Relatively few genes (13) had an increased transcript level in both salt-stressed genotypes and about half of them were functionally related to stress response (Table 1). The tran-scripts of glutathione reductase (GR) and pheophorbide a oxy-genase, were more induced in 14P11 than in 6K3. Glutathione reductase is an enzyme of the ascorbate–glutathione cycle that contributes to the defense against ROS by maintaining glutathione in the reduced state (GSH) (Gill and Tuteja 2010). The expression of GR genes in plants is induced by abiotic stresses and particularly by heavy metals. A genotype- specific induction of the ROS-scavenging pathway may lead to better tolerance of abiotic stresses. Therefore, the great induction of GR genes in 14P11 could be linked to adaptation as a result of environmental selection pressure exerted by the original habitat of this genotype. Supporting this hypothesis, a differential expression of GR genes was observed between two Populus nigra L. genotypes originating from North and South Italy after exposure to water deficit. GR transcripts increased in the southern genotypes but not in the northern genotype (Regier et al. 2009). The second salinity-induced gene, pheophorbide a oxygenase (PAO), encodes a thiore-doxin involved in chlorophyll breakdown and protein translo-cation across chloroplast membranes (Bartsch et al. 2008). This protein degrades pheophorbide a, which is a toxic interme-diate of chlorophyll catabolism. The accumulation of pheophor-bide a causes cell death in transgenic Arabidopsis plants with antisense silencing of PAO genes. (Hirashima et al. 2009). In P. alba plants, PAO induction could mitigate the increase of pheophorbide a originating from chlorophyll degradation under salinity stress.

The transcripts of three ESTs homologous to a zinc finger (C3HC4 ring finger) protein also increased more markedly in 14P11 than in 6K3 after salt stress. The C3HC4 ring finger protein is involved in the ubiquitin-dependent protein degrada-tion by the 26S proteasome and targets many protein sub-strates such as receptors, nuclear transcription regulators, abnormal proteins and other short-lived regulatory proteins (Stone et al. 2005). This class of ring finger protein is induced by abiotic stresses and was shown to be up-regulated in plants after exposure to cadmium (Minglin et al. 2005, Zeba et al. 2006). The differential expression of this gene in salt-stressed P. alba leaves could be related to increased protein turnover and re-modeling of the leaf proteome.

Two ESTs homologous to bark storage proteins (BSPs) were more induced in 6K3 than in 14P11 after salt stress. BSPs are the main form of nitrogen reserve in Populus and are translo-cated from senescing leaves in autumn to supply reduced nitrogen for spring growth. A wide genetic variability for the expression of genes encoding BSP has been reported within Populus species (Black et al. 2001). The stronger increase in the expression of these genes in 6K3 might indicate incipient leaf senescence induced by salt stress.

Gene regulation by salinity: genotype-specific genes

Most 6K3-specific genes had a reduced transcript level and their expression in 14P11 was just below the threshold of two-fold change (Table 3). The most pronounced reduction was observed in key genes of phenylpropanoid–flavonoid pathway, especially phenylalanine ammonia-lyase and flavanone 3-hydrox-ylase. Flavonoids and other polyphenols are strong antioxidants, thus contributing to stress tolerance (Rice-Evans et al. 1997). Moreover, a higher content of phenolic compounds was observed in leaves of P. euphratica (salt tolerant) than in those of P. canescens Sm. (salt sensitive) (Janz et al. 2010). The decreased expression of genes from the phenyl–propanoid pathway in 6K3 could enhance the negative impact of salinity stress and explain the sensitivity of this genotype to salt stress.

Only a few ESTs with increased expression were specific to 6K3. The most interesting of these genes is late embryogenesis abundant (LEA) (EST AJ779923). The BlastX analysis of this EST found homology to LEA group 5. Experimental evidence indicates that LEA proteins are involved in desiccation toler-ance and that over expression of LEA increases drought toler-ance in transgenic plants. The mechanisms of action are still unclear but it seems that LEAs protect proteins against dena-turation (Khurana et al. 2008). The differential accumulation of LEAs in response to salt stress was already observed in leaves of two Morus alba L. genotypes with contrasting salinity toler-ance, as some LEA isoforms were up-regulated in the tolerant genotype and down-regulated in the salt-sensitive genotype (Jyothsnakumari et al. 2009). In the present study, the increased expression of LEA 5 genes was found in the salt-sensitive 6K3. Additional physiological analyses to assess the occurrence of the osmotic component of the stress in leaves could help in explaining this result and further studies on expression of LEA genes should investigate the different LEA isoforms.

A few transcripts were specifically salt-decreased in 14P11 (Table 2), but their functional annotation addressed the bio-logical processes that were also found in genes with decreased expression of both genotypes (Table 1). Therefore, these genes do not discriminate the salt-responsive transcrip-tome of the two genotypes. In this group, only the gene AP2 ERF domain-containing transcription factor showed a strong decrease of transcripts in 14P11. The down-regulation of this gene in response to water deficit was already observed in the

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cambial region of P. alba clone Marte (Berta et al. 2010), which is considered salinity tolerant (Salvati et al. 2007). In contrast, Nicotiana tabacum L. plants over expressing this gene demonstrated more tolerance to dehydration and salt stress than the wild-type plants (Shukla et al. 2006). AP2 ERF regulates multiple processes of plant development and metabolism, including secondary metabolism, responses to biotic and abiotic stress, and responses to different hor-mones. Based only on the existing evidence, we cannot say why the transcriptional regulation of AP2 ERF appears to be opposite in Nicotiana and Populus. However, as the down-regulation of AP2 ERF also occurs in other salt-tolerant P. alba genotypes in response to stress, this transcription factor can be considered as a candidate for response to abiotic stresses in this species.

In contrast to 6K3, the genes specifically salinity-regulated in 14P11 mostly had increased expression and cover specific biological processes that discriminate the two genotypes. One of these processes is ion transport across membranes. The expression of P-type proton pump and V-type proton pump genes was specifically increased by salinity in 14P11 plants. These proteins can drive transport across membranes, includ-ing active exclusion of Na+ from the cytoplasm, as has been discussed above. The specific induction of these genes could contribute to the better tolerance of 14P11 under long-term salinity.

Osmolyte biosyntheses were also significantly induced only in 14P11 through an increased expression of two genes: trehealose-6-phosphate synthase and asparagine synthetase (AS). The first gene encodes a key enzyme for the biosynthesis of trehalose, which can act as a compatible osmolyte for osmotic adjustment and as an osmoprotectant. In transgenic plants, the over expression of this gene improved stress toler-ance and protected the photosynthetic apparatus against heat damage (Jun et al. 2008, Santamaria et al. 2009). The AS gene encodes a key enzyme for the synthesis of asparagine, a key nitrogen transporter in plants. An increased level of AS tran-scripts was previously observed in plants in response to salinity and other abiotic stresses (Wang et al. 2005, Herrera-Rodríguez et al. 2007). Asparagine synthetase enzyme transforms gluta-mine into asparagine by transfer of the amino group, but there is some evidence that AS can use ammonia directly. In this way the accumulation of asparagine could serve as a detoxification mechanism to reduce the excess of ammonia deriving from stress-induced protein catabolism and photorespiration (Lea and Azevedo 2007). It should be noted that the expression of glutamine synthetase (GS) genes decreased specifically in salt-stressed plants of 14P11. Glutamine synthetase activity can also use ammonium deriving from protein catabolism as a sub-strate to form glutamine (Bernard and Habash 2009). The opposite regulation of AS and GS genes suggests a salt-stress modulation of nitrogen metabolism, favoring an accumulation of

asparagine. This shift could have positive implications for a more efficient nitrogen recycling in leaves, since asparagine has a high C:N ratio (Lea and Azevedo 2007).

Two more genes related to stress response were specifically induced by salinity in 14P11: heat shock protein 70 kDa (HSP70) and osmotin-like protein. Heat shock protein 70 has chaperone functions, prevents protein aggregation and assists protein refolding, protein import and translocation into organ-elles. It facilitates the degradation of unstable proteins by tar-geting the proteins to lysosomes or proteasomes. Moreover, HSP70 protein regulates signal transduction and transcrip-tional activation by interacting with heat shock factors (Wang et al. 2004). Heat shock protein 70 appears as an interesting candidate gene for salinity tolerance in P. alba, but functional studies are required to clarify its mechanisms of action in this species. Osmotins are pathogen-related proteins that are induced by many biotic and abiotic stresses and their synthesis is regulated by ABA. The same osmotin gene (EST AJ779386) was also stress induced in leaves and roots of P. euphratica after 1 day of exposure to salinity (Brinker et al. 2010). Experimental evidence indicates that osmotin is involved in response to osmotic stress (Singh et al. 1987). In Capsicum annuum L. (Subramanyam et al. 2010) and in Morus indica L. (Das et al. 2010), overexpression of osmotin conferred better tolerance to drought and salinity, with increases in proline, osmoprotectants and ROS detoxification enzymes. Probably the role of osmotin in P. alba leaves is also related to cell protection against osmotic stress.

Two ESTs encoding polyphenol oxidase (PPO) had strongly increased expression in 14P11. Mechanical wounding and her-bivore attacks induce PPO in poplars (Constabel et al. 2000), and the involvement of PPO in response to drought and salinity has also been reported in several plant species, but the under-lying mechanisms are not yet clear (Mayer 2006). Polyphenols are the substrate of PPO activity and their protective role as antioxidants in the stress-tolerance mechanism has been dis-cussed above (Rice-Evans et al. 1997). A decrease in PPO activity was observed in the leaves of olive trees subjected to salinity (Ben Ahmed et al. 2010). The antisense suppression of PPO made tomato plants more tolerant to drought (Thipyapong et al. 2004). It has been proposed that down-regulation of PPO is required for an efficient adaptation to salinity and osmotic stress, because more polyphenols are then available as anti-oxidants. Based on these facts, the strong induction of PPO expression in 14P11 could be interpreted as a disadvantage for salinity tolerance. It is interesting that the structural genes of the phenylpropanoid biosynthetic pathway (PAL, CHS, FL3H and DHFR) were less affected by salinity in 14P11 than in 6K3. The regulation of these pathways could be interlinked, but fur-ther investigation of PPO enzymatic activity and content of phenolic compounds is needed to clarify the role of secondary metabolites in salinity tolerance.

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Conclusion

Recent studies point to many genes that mediate salt tolerance in several plant species. A better knowledge of natural variation of this trait in Populus, as well as functional analysis of candi-date genes, will help in understanding the physiological and molecular basis of salinity tolerance and, ultimately, will con-tribute to the improvement of tree productivity. In this study, a transcriptome analysis of two well-characterized P. alba geno-types that differ in salt tolerance has provided new information for the identification of genetic determinants of salinity toler-ance. In the short term, acute salinity stress strongly inhibited the physiological functionality of leaves in both genotypes. The decreased expression of carbonic anhydrase, rubisco subunits, rubisco activase and plastidic aldolase genes helps to explain the inhibition of photosynthesis. However, the comparative analysis of transcriptome revealed differences in gene expres-sion and molecular mechanisms that confer diverse tolerance to these genotypes. By subtracting the common transcriptome changes induced by salinity, this comparative approach allowed us to identify key salinity-responsive genes linked to the natu-ral variation of transcriptome within P. alba species. Several genes, specifically induced in genotype 14P11, were involved in ion transport (P-type proton pump and V-type proton pump), osmolyte biosynthesis (trehalose-6-phosphate synthase and AS), stress response (osmolyte biosynthesis, HSP70 and osmotin-like protein) and secondary metabolism (polyphenol oxidase). On the contrary, genotype 6K3 showed decreased expression of genes related to secondary metabolism (phenyl-alanine ammonia-lyase and flavanone 3-hydroxylase) and one interesting gene, LEA, functionally involved in desiccation toler-ance, was specifically induced in this genotype. This could be the result of natural selection for adaptation to contrasting habitats, which differ in climate features. The genes and bio-logical processes specifically induced in the southern geno-type 14P11 represent key mechanisms for salinity tolerance and may be related to the stressful conditions of its natural habitat. The stress-responsive genes identified here are valu-able resources for further functional genomics studies address-ing tolerance to salinity in poplar species. A more complete picture of tolerance mechanisms in Populus will be obtained by RNA-Seq technology, which using next-generation deep sequencing provides a robust comparison of levels of tran-scripts and their isoforms to unravel gene networks and regula-tory mechanisms of stress tolerance in trees. In addition, since the proteins encoded by these genes have not been analyzed, the post-transcriptional level of regulation remains to be eluci-dated in future proteomics studies. Salinity tolerance is a com-plex multigenic trait, and thus more insights might be gained by combining quantitative trait locus (QTL) mapping with tran-scriptome and proteome analysis. The availability of a mapping pedigree and genetic maps (Paolucci et al. 2010) from the

cross of P. alba genotypes 14P11 and 6K3 would allow such an approach. The co-localization of QTL regions with the genome position of functional candidate genes would greatly empower the identification of key genes for salinity tolerance.

Supplementary data

Supplementary data for this article are available at Tree Physiology Online.

Acknowledgments

The authors would like to thank Dr Paola Pinelli (National Research Council, Italy) for the ecophysiology analyses; Prof. Alessio Valentini (University of Tuscia, Italy) for access to the microarray facilities; Mrs Matilde Tamantini (University of Tuscia, Italy) for technical assistance; and Dr Guido Bongi (National Research Council, Italy) for critical comments on the ecophysiology results. Turku Centre for Biotechnology, University of Turku and Åbo Akademi University (Finland) are acknowledged for providing the arrays.

Funding

This work was supported by the European Community’s Fifth Framework Program (POPYOMICS QLK5-CT-2001-00953); the Italian Ministry of the Environment and Territories project ‘Italy-Israel Cooperation on Environmental research and devel-opment’; the Italian Ministry of Education, University and Research (Brain Gain Program ‘Rientro dei cervelli’ to A.H.).

References

Abbruzzese, G., I. Beritognolo, R. Muleo, M. Piazzai, M. Sabatti, G. Scarascia Mugnozza and E. Kuzminsky. 2009. Leaf morphological plasticity and stomatal conductance in three Populus alba L. geno-types subjected to salt stress. Environ. Exp. Bot. 66:381–388.

Altschul, S.F., T.L. Madden, A.A. Schaffer, J. Zhang, Z. Zhang, W. Miller and D. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new genera-tion of protein database search programs. Nucleic Acids Res. 25:3389–3402.

Bartsch, S., J. Monnet, K. Selbach, F. Quigley, J. Gray, D. Von Wettstein, S. Reinbothe and C. Reinbothe. 2008. Three thioredoxin targets in the inner envelope membrane of chloroplasts function in protein import and chlorophyll metabolism. Proc. Natl Acad. Sci. USA 105:4933–4938.

Ben Ahmed, C., B. Ben Rouina, S. Sensoy, M. Boukhriss and F. Ben Abdullah. 2010. Exogenous proline effects on photosynthetic per-formance and antioxidant defense system of young olive tree. J. Agric. Food Chem. 58:4216–4222.

Benjamini, Y. and Y. Hochberg. 1995. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. R. Stat. Soc. B 57:289–300.

Beritognolo, I., M. Piazzai, S. Benucci, E. Kuzminsky, M. Sabatti, G. Scarascia Mugnozza and R. Muleo. 2007. Functional characterisa-tion of three Italian Populus alba L. genotypes under salinity stress. Trees-Struct. Funct. 21:465–477.

1352 Beritognolo et al.

at National L

ibrary of Health Sciences on M

arch 7, 2012http://treephys.oxfordjournals.org/

Dow

nloaded from

Tree Physiology Online at http://www.treephys.oxfordjournals.org

Bernard, S.M. and D.Z. Habash. 2009. The importance of cytosolic glutamine synthetase in nitrogen assimilation and recycling. New Phytol. 182:608–620.

Berta, M., A. Giovannelli, F. Sebastiani, A. Camussi and M.L. Racchi. 2010. Transcriptome changes in the cambial region of poplar (Populus alba L.) in response to water deficit. Plant Biol. 12: 341–354.

Black, B.L., C.M. Parmentier-Line, L.H. Fuchigami and G.D. Coleman. 2001. Ecotypic and genetic variation in poplar bark storage protein gene expression and accumulation. Tree Physiol. 21:1289–1297.

Blumwald, E. 2000. Sodium transport and salt tolerance in plants. Curr. Opin. Cell Biol. 12:432–434.

Blumwald, E., G.S. Aharon and M.P. Apse. 2000. Sodium transport in plant cells. Biochim. Biophys. Acta 1465:140–151.

Brinker, M., M. Brosche, B. Vinocour, et al. 2010. Linking the salt tran-scriptome with physiological responses of a salt resistant Populus species as a strategy to identify genes important for stress acclima-tion. Plant Physiol. 154:1697–1709.

Brosché, M., B. Vinocur, E.R. Alatalo, et al. 2005. Gene expression and metabolite profiling of Populus euphratica growing in the Negev desert. Genome Biol. 6:R101.

Brunner, A.M., I.A. Yakovlev and S.H. Strauss. 2004. Validating internal controls for quantitative plant gene expression studies. BMC Plant Biol. 4:14.

Chang, S., J. Puryear and J. Cairney. 1993. A simple and efficient method for isolating RNA from pine trees. Plant Mol. Biol. Rep. 11:113–116.

Chaves, M.M., J. Flexas and C. Pinheiro. 2009. Photosynthesis under drought and salt stress: regulation mechanisms from whole plant to cell. Ann. Bot. 103:551–560.

Chelaifa, H., F. Mahé and M. Ainouche. 2010. Transcriptome diver-gence between the hexaploid salt-marsh sister species Spartina maritima and Spartina alterniflora (Poaceae). Mol. Ecol. 19:2050–2063.

Chen, S. and A. Polle. 2010. Salinity tolerance of Populus. Plant Biol. 12:317–333.

Chen, S., J. Li, S. Wang, A. Hüttermann and A. Altman. 2001. Salt, nutri-ent uptake and transport, and ABA of Populus euphratica; a hybrid in response to increasing soil NaCl. Trees-Struct. Funct. 15:186–194.

Chen, S., J. Li, E. Fritz, S. Wang and A. Hüttermann. 2002. Sodium and chloride distribution in roots and transport in three poplar genotypes under increasing NaCl stress. For. Ecol. Manag. 168:217–230.

Chen, S., J. Li, S. Wang, E. Fritz, A. Hüttermann and A. Altman. 2003. Effects of NaCl on shoot growth, transpiration, ion compartmenta-tion, and transport in regenerated plants of Populus euphratica and Populus tomentosa. Can. J. For. Res. 33:967–975.

Cocozza, C., A. Minnocci, R. Tognetti, V. Iori, M. Zacchini and G. Scarascia Mugnozza. 2008. Distribution and concentration of cad-mium in root tissue of Populus alba determined by scanning electron microscopy and energy-dispersive x-ray microanalysis. iForest 1:96–103.

Conesa, A., S. Götz, J.M. García-Gómez, J. Terol, M. Talón and M. Robles. 2005. Blast2GO: a universal tool for annotation, visualiza-tion and analysis in functional genomics research. Bioinformatics 21:3674–3676.

Constabel, C.P., L. Yip, J.J. Patton and M.E. Christopher. 2000. Polyphenol oxidase from hybrid poplar. Cloning and expression in response to wounding and herbivory. Plant Physiol. 124:285–295.

Das, M., H. Chauhan, A. Chhibbar, Q.M. Rizwanul Haq and P. Khurana. 2010. High-efficiency transformation and selective tolerance against biotic and abiotic stress in mulberry, Morus indica cv. K2, by consti-tutive and inducible expression of tobacco osmotin. Transgenic Res. 20:231–246.

Delfine, S., A. Alvino, M.C. Villani and F. Loreto. 1999. Restriction to carbon dioxide conductance and photosynthesis in spinach leaves recovering from salt stress. Plant Physiol. 119:1101–1106.

Deyholos, M.K. 2010. Making the most of drought and salinity tran-scriptomics. Plant Cell Environ. 33:648–654.

Di Martino, C., S. Delfine, R. Pizzuto, F. Loreto and A. Fuggi. 2003. Free amino acids and glycine betaine in leaf osmoregulation of spinach responding to increasing salt stress. New Phytol. 158:455–463.

Ding, M., P. Hou, X. Shen, et al. 2010. Salt-induced expression of genes related to Na+/K+ and ROS homeostasis in leaves of salt- resistant and salt-sensitive poplar species. Plant Mol. Biol. 73:251–269.

Ergen, N.Z., J. Thimmapuram, H.J. Bohnert and H. Budak. 2009. Transcriptome pathways unique to dehydration tolerant relatives of modern wheat. Funct. Integr. Genomics 9:377–396.

Fang, Y., A. Brass, D.C. Hoyle, A. Hayes, A. Bashein, S.G. Oliver, D. Waddington and M. Rattray. 2003. A model-based analysis of microarray experimental error and normalisation. Nucleic Acids Res. 31:e96.

Flexas, J., J. Bota, F. Loreto, G. Cornic and T.D. Sharkey. 2004. Diffusive and metabolic limitations to photosynthesis under drought and salinity in C3 plants. Plant Biol. 6:269–279.

Gill, S.S. and N. Tuteja. 2010. Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol. Biochem. 48:909–930.

Gillon, J.S. and D. Yakir. 2000. Internal conductance to CO2 diffusion and C18OO discrimination in C3 leaves. Plant Physiol. 123: 201–214.

Gong, Q., P. Li, S. Ma, S. Indu Rupassara and H.J. Bohnert. 2005. Salinity stress adaptation competence in the extremophile Thellungiella halophila in comparison with its relative Arabidopsis thaliana. Plant J. 44:826–839.

Herrera-Rodríguez, M.B., R. Pérez-Vicente and J.M. Maldonado. 2007. Expression of asparagine synthetase genes in sunflower (Helianthus annuus) under various environmental stresses. Plant Physiol. Biochem. 45:33–38.

Hirashima, M., R. Tanaka and A. Tanaka. 2009. Light-independent cell death induced by accumulation of pheophorbide a in Arabidopsis thaliana. Plant Cell Physiol. 50:719–729.

Horie, T., F. Hauser and J.I. Schroeder. 2009. HKT transporter-medi-ated salinity resistance mechanisms in Arabidopsis and monocot crop plants. Trends Plant Sci. 14:660–668.

Imada, S., N. Yamanaka and S. Tamai. 2009. Effects of salinity on the growth, Na partitioning, and Na dynamics of a salt-tolerant tree, Populus alba L. J. Arid Environ. 73:245–251.

Janz, D., K. Behnke, J.P. Schnitzler, B. Kanawati, P. Schmitt-Kopplin and A. Polle. 2010. Pathway analysis of the transcriptome and metabo-lome of salt sensitive and tolerant poplar species reveals evolution-ary adaption of stress tolerance mechanisms. BMC Plant Biol. 10:150.

Jun, S.S., J.C. Hye, Y.L. Hae and Y.N. Hong. 2008. Differential protec-tion of photosynthetic capacity in trehalose- and LEA protein-pro-ducing transgenic plants under abiotic stresses. J. Plant Biol. 51:327–336.

Jyothsnakumari, G., M. Thippeswamy, G. Veeranagamallaiah and C. Sudhakar. 2009. Differential expression of LEA proteins in two geno types of mulberry under salinity. Biol. Plantarum 53:145–150.

Katiyar-Agarwal, S., J. Zhu, K. Kim, M. Agarwal, X. Fu, A. Huang and J.K. Zhu. 2006. The plasma membrane Na+/H+ antiporter SOS1 inter-acts with RCD1 and functions in oxidative stress tolerance in Arabidopsis. Proc. Natl Acad. Sci. USA 103:18816–18821.

Khurana, P., D. Vishnudasan and A.K. Chhibbar. 2008. Genetic approaches towards overcoming water deficit in plants - special emphasis on LEAs. Physiol. Mol. Biol. Plants 14:277–298.

Transcriptome response to salinity in Populus alba 1353

at National L

ibrary of Health Sciences on M

arch 7, 2012http://treephys.oxfordjournals.org/

Dow

nloaded from

Tree Physiology Volume 31, 2011

Lea, P.J. and R.A. Azevedo. 2007. Nitrogen use efficiency. 2. Amino acid metabolism. Ann. Appl. Biol. 151:269–275.

Loreto, F., M. Centritto and K. Chartzoulakis. 2003. Photosynthetic limitations in olive cultivars with different sensitivity to salt stress. Plant Cell Environ. 26:595–601.

Ma, X., L. Deng, J. Li, et al. 2010. Effect of NaCl on leaf H+-ATPase and the relevance to salt tolerance in two contrasting poplar species. Trees-Struct. Funct. 24:597–607.

Mane, S.P., C.V. Robinet, A. Ulanov, et al. 2008. Molecular and physio-logical adaptation to prolonged drought stress in the leaves of two Andean potato genotypes. Funct. Plant. Biol. 35:669–688.

Mao, H., F. Iwanaga, N. Yamanaka and F. Yamamoto. 2008. Growth, photosynthesis, and ion distribution in hydroponically cultured Populus alba L. cuttings grown under various salinity concentrations. Landsc. Ecol. Eng. 4:75–82.

Mao, H., Y. Okada, S. Michimata, W. Wang, F. Iwanaga, N. Yamanaka and F. Yamamoto. 2010. Responses to nonaeration and/or salinity stress in hydroponically cultured Populus nigra and Populus alba cuttings. Landsc. Ecol. Eng. 6:11–21.

Marron, N., L. Ricciotti, C. Bastien, et al. 2011. Plasticity of growth and biomass production of an intraspecific Populus alba family grown at three sites across Europe during three growing seasons. Can. J. For. Res. 40:1887–1903.

Mayer, A.M. 2006. Polyphenol oxidases in plants and fungi: going places? A review. Phytochemistry 67:2318–2331.

Mehta, P., A. Jajoo, S. Mathur and S. Bharti. 2010. Chlorophyll a fluo-rescence study revealing effects of high salt stress on photosystem II in wheat leaves. Plant Physiol. Biochem. 48:16–20.

Minglin, L., Z. Yuxiu and C. Tuanyao. 2005. Identification of genes up-regulated in response to Cd exposure in Brassica juncea L. Gene 363:151–158.

Mohammadi, M., N.N.V. Kav and M.K. Deyholos. 2008. Transcript expression profile of water-limited roots of hexaploid wheat (Triticum aestivum ‘Opata’). Genome 51:357–367.

Moroney, J.V., S.G. Bartlett and G. Samuelsson. 2001. Carbonic anhy-drases in plants and algae. Plant Cell Environ. 24:141–153.

Munns, R. and M. Tester. 2008. Mechanisms of salinity tolerance. Annu. Rev. Plant Biol. 59:651–681.

Munns, R., R.A. James and A. Lauchli. 2006. Approaches to increasing the salt tolerance of wheat and other cereals. J. Exp. Bot. 57:1025–1043.

Murata, N., S. Takahashi, Y. Nishiyama and S.I. Allakhverdiev. 2007. Photoinhibition of photosystem II under environmental stress. BBA-Bioenergetics 1767:414–421.

Nishiyama, Y., S.I. Allakhverdiev and N. Murata. 2006. A new paradigm for the action of reactive oxygen species in the photoinhibition of photosystem II. BBA-Bioenergetics 1757:742–749.

Oh, D.H., M. Dassanayake, J.S. Haas, et al. 2010. Genome structures and halophyte-specific gene expression of the extremophile Thellungiella parvula in comparison with Thellungiella salsuginea (Thellungiella halophila) and arabidopsis. Plant Physiol. 154:1040–1052.

Ottow, E.A., A. Polle, M. Brosché, J. Kangasjarvi, P. Dibrov, C. Zörb and T. Teichmann. 2005a. Molecular characterization of PeNhaD1: the first member of the NhaD Na+/H+ antiporter family of plant origin. Plant Mol. Biol. 58:75–88.

Ottow, E.A., M. Brinker, T. Teichmann, E. Fritz, W. Kaiser, M. Brosché, J. Kangasjarvi, X. Jiang and A. Polle. 2005b. Populus euphratica displays apoplastic sodium accumulation, osmotic adjustment by decreases in calcium and soluble carbohydrates, and develops leaf succulence under salt stress. Plant Physiol. 139:1762–1772.

Paolucci, I., M. Gaudet, V. Jorge, I. Beritognolo, S. Terzoli, E. Kuzminsky, R. Muleo, G.S. Mugnozza and M. Sabatti. 2010. Genetic linkage maps of Populus alba L. and comparative mapping analysis of sex

determination across Populus species. Tree Genet. Genomes 6:863–875.

Pfaffl, M.W., G.W. Horgan and L. Dempfle. 2002. Relative expression soft-ware tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res. 30:e36.

R Development Core Team. 2010. R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria.

Ramakers, C., J.M. Ruijter, R.H. Lekanne Deprez and A.F.M. Moorman. 2003. Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neurosci. Lett. 339:62–66.

Regier, N., S. Streb, C. Cocozza, M. Schaub, P. Cherubini, S.C. Zeeman and B. Frey. 2009. Drought tolerance of two black poplar (Populus nigra L.) clones: contribution of carbohydrates and oxidative stress defence. Plant Cell Environ. 32:1724–1736.

Reiner, A., D. Yekutieli and Y. Benjamini. 2003. Identifying differentially expressed genes using false discovery rate controlling procedures. Bioinformatics 19:368–375.

Rice-Evans, C.A., N.J. Miller and G. Paganga. 1997. Antioxidant proper-ties of phenolic compounds. Trends Plant Sci. 2:152–159.

Rodrigues, F.A., M.L. de Laia and S.M. Zingaretti. 2009. Analysis of gene expression profiles under water stress in tolerant and sensitive sugarcane plants. Plant Sci. 176:286–302.

Roshandel, P. and T. Flowers. 2009. The ionic effects of NaCl on physi-ology and gene expression in rice genotypes differing in salt toler-ance. Plant Soil 315:135–147.

Rozen, S. and H. Skaletsky. 2000. Primer3 on the WWW for general users and for biologist programmers. Methods Mol. Biol. 132:365–386.

Rus, A.M., R.A. Bressan and P.M. Hasegawa. 2005. Unraveling salt tolerance in crops. Nat. Genet. 37:1029–1030.

Sabatti, M., R. D’Ovidio, O.A. Tanzarella and G.E. Scarascia Mugnozza. 2001. Assessment of geographic variation by RAPD markers among Italian open-pollinated progenies of Populus alba L. Genet. Resour. Crop Ev. 48:423–428.

Saeed, A.I., V. Sharov, J. White, et al. 2003. TM4: A free, open-source system for microarray data management and analysis. Biotechniques 34:374–378.

Sakamoto, A. and N. Murata. 2002. The role of glycine betaine in the protection of plants from stress: clues from transgenic plants. Plant Cell Environ. 25:163–171.

Salvati, R., G. Chirici and P. Corona. 2007. Modello di valutazione dell’attitudine fisica del territorio per la realizzazione di impianti cedui da biomassa in Italia. Italia Forest. Mont. 62:399–410.

Santamaria, J.M., D. Hernasndez-Portilla, B. Chi-Manzanero, F. Espadas, E. Castaño, G. Iturriaga and L.C. Rodriguez-Zapata. 2009. Incorporation of two trehalose biosynthetic genes in banana increases trehalose levels and protects the photosyn-thetic apparatus from salt-stress damage. J. Hortic. Sci. Biotech. 84:665–671.

Scharpf, R.B., C.A. Iabocuzio-Donahue, J.B. Sneddon and G. Parmigiani. 2007. When should one substract background fluorescence in two color microarrays? Biostatistics 8:695–707.

Shi, H., M. Ishitani, C. Kim and J.K. Zhu. 2000. The Arabidopsis thaliana salt tolerance gene SOS1 encodes a putative Na+/H+ antiporter. Proc. Natl Acad. Sci. USA 97:6896–6901.

Shukla, R.K., S. Raha, V. Tripathi and D. Chattopadhyay. 2006. Expression of CAP2, an APETALA2-family transcription factor from chickpea, enhances growth and tolerance to dehydration and salt stress in transgenic tobacco. Plant Physiol. 142:113–123.

Singh, N.K., C.A. Bracker, P.M. Hasegawa, A.K. Handa, S. Buckel, M.A. Hermodson, E. Pfankoch, F.E. Regnier and R.A. Bressan. 1987. Characterization of osmotin: a thaumatin-like protein associated with osmotic adaptation in plant cells. Plant Physiol. 85:529–536.

1354 Beritognolo et al.

at National L

ibrary of Health Sciences on M

arch 7, 2012http://treephys.oxfordjournals.org/

Dow

nloaded from

Tree Physiology Online at http://www.treephys.oxfordjournals.org

Sixto, H., J.M. Grau, N. Alba and R. Alia. 2005. Response to sodium chloride in different species and clones of genus Populus L. Forestry 78:93–104.

Sixto, H., I. Aranda and J.M. Grau. 2006. Assessment of salt tolerance in Populus alba clones using chlorophyll fluorescence. Photosynthetica 44:169–173.

Stone, S.L., H. Hauksdóttir, A. Troy, J. Herschleb, E. Kraft and J. Callis. 2005. Functional analysis of the RING-type ubiquitin ligase family of Arabidopsis. Plant Physiol. 137:13–30.

Subramanyam, K., K.V. Sailaja, K. Subramanyam, D. Muralidhara Rao and K. Lakshmidevi. 2010. Ectopic expression of an osmotin gene leads to enhanced salt tolerance in transgenic chilli pepper (Capsicum annuum L.). Plant Cell Tissue Organ Cult. 105:181–192.

Sun, J., M.J. Wang, M.Q. Ding, et al. 2010a. H2O2 and cytosolic Ca2+ signals triggered by the PM H+-coupled transport system mediate K+/Na+ homeostasis in NaCl-stressed Populus euphratica cells. Plant Cell Environ. 33:943–958.

Sun, W., X. Xu, H. Zhu, A. Liu, L. Liu, J. Li and X. Hua. 2010b. Comparative transcriptomic profiling of a salt-tolerant wild tomato species and a salt-sensitive tomato cultivar. Plant Cell Physiol. 51:997–1006.

Taji, T., M. Seki, M. Satou, et al. 2004. Comparative genomics in salt tolerance between arabidopsis and arabidopsis-related halophyte salt cress using arabidopsis microarray. Plant Physiol. 135:1697–1709.

Takahashi, S. and N. Murata. 2008. How do environmental stresses accelerate photoinhibition? Trends Plant Sci. 13:178–182.

Tang, R.J., H. Liu, Y. Bao, Q.D. Lv, L. Yang and H.X. Zhang. 2010. The woody plant poplar has a functionally conserved salt overly sensi-tive pathway in response to salinity stress. Plant Mol. Biol. 74:367–380.

Tester, M. and R. Davenport. 2003. Na+ tolerance and Na+ transport in higher plants. Ann. Bot. 91:503–527.

Thipyapong, P., J. Melkonian, D.W. Wolfe and J.C. Steffens. 2004. Suppression of polyphenol oxidases increases stress tolerance in tomato. Plant Sci. 167:693–703.

Vinocur, B. and A. Altman. 2005. Recent advances in engineering plant tolerance to abiotic stress: achievements and limitations. Curr. Opin. Biotechnol. 16:123–132.

Walia, H., C. Wilson, P. Condamine, et al. 2005. Comparative tran-scriptional profiling of two contrasting rice genotypes under salinity stress during the vegetative growth stage. Plant Physiol. 139:822–835.

Wang, W., B. Vinocur, O. Shoseyov and A. Altman. 2004. Role of plant heat-shock proteins and molecular chaperones in the abiotic stress response. Trends Plant Sci. 9:244–252.

Wang, H., D. Liu, J. Sun and A. Zhang. 2005. Asparagine synthetase gene TaASN1 from wheat is up-regulated by salt stress, osmotic stress and ABA. J. Plant Physiol. 162:81–89.

Wang, R., S. Chen, L. Deng, E. Fritz, A. Hüttermann and A. Polle. 2007. Leaf photosynthesis, fluorescence response to salinity and the rel-evance to chloroplast salt compartmentation and anti-oxidative stress in two poplars. Trees-Struct. Funct. 21:581–591.

Wu, Y., N. Ding, X. Zhao, M. Zhao, Z. Chang, J. Liu and L. Zhang. 2007. Molecular characterization of PeSOS1: the putative Na(+)/H (+) anti-porter of Populus euphratica. Plant Mol. Biol. 65:1–11.

Yamada, S., T. Komori, A. Hashimoto, S. Kuwata, H. Imaseki and T. Kubo. 2000. Differential expression of plastidic aldolase genes in Nicotiana plants under salt stress. Plant Sci. 154:61–69.

Yang, Y.H., S. Dudoit, P. Luu and T.P. Speed. 2001. Normalization for cDNA microarray data. In Microarrays: Optical Technologies and Informatics. Eds. M. Bittner, Y. Chen, A. Dorsel and E.R. Dougherty. SPIE, San Jose, CA, pp 141–152.

Zeba, N., M. Ashrafuzzaman and B.H. Choo. 2006. Molecular charac-terization of the Capsicum annuum RING zinc finger protein 1 (CaRZFP1) gene induced by abiotic stresses. J. Plant Biol. 49:484–490.

Zhu, J.K. 2002. Salt and drought stress signal transduction in plants. Annu. Rev. Plant Biol. 53:247–273.

Zhu, J.K. 2003. Regulation of ion homeostasis under salt stress. Curr. Opin. Plant Biol. 6:441–445.

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