Transcriptome differences between two sister desert poplar species under salt stress
© Zhang et al.; licensee BioMed Central Ltd. 2014
Received: 13 July 2013
Accepted: 30 April 2014
Published: 4 May 2014
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© Zhang et al.; licensee BioMed Central Ltd. 2014
Received: 13 July 2013
Accepted: 30 April 2014
Published: 4 May 2014
Populus euphratica Oliv and P. pruinosa Schrenk (Salicaceae) both grow in dry desert areas with high summer temperatures. However, P. euphratica is distributed in dry deserts with deep underground water whereas P. pruinosa occurs in deserts in which there is underground water close to the surface. We therefore hypothesized that these two sister species may have evolved divergent regulatory and metabolic pathways during their interaction with different salt habitats and other stresses. To test this hypothesis, we compared transcriptomes from callus exposed to 24 h of salt stress and control callus samples from both species and identified differentially expressed genes (DEGs) and alternative splicing (AS) events that had occurred under salt stress.
A total of 36,144 transcripts were identified and 1430 genes were found to be differentially expressed in at least one species in response to salt stress. Of these DEGs, 884 and 860 were identified in P. euphratica and P. pruinosa, respectively, while 314 DEGs were common to both species. On the basis of parametric analysis of gene set enrichment, GO enrichment in P. euphratica was found to be significantly different from that in P. pruinosa. Numerous genes involved in hormone biosynthesis, transporters and transcription factors showed clear differences between the two species in response to salt stress. We also identified 26,560 AS events which were mapped to 8380 poplar genomic loci from four libraries. GO enrichments for genes undergoing AS events in P. euphratica differed significantly from those in P. pruinosa.
A number of salt-responsive genes in both P. euphratica and P. pruinosa were identified and candidate genes with potential roles in the salinity adaptation were proposed. Transcriptome comparisons of two sister desert poplar species under salt stress suggest that these two species may have developed different genetic pathways in order to adapt to different desert salt habitats. The DEGs that were found to be common to both species under salt stress may be especially important for future genetic improvement of cultivated poplars or other crops through transgenic approaches in order to increase tolerance of saline soil conditions.
Salinity and drought stresses are the two most important environmental factors limiting plant growth and development in semiarid and arid areas . Over 100 countries in the world have been identified as being affected by salinity , and the scale of the problem seems to be increasing at an alarming rate . Salinity, together with drought, has far-reaching implications for food security, economic sustainability and the irreplaceable biodiversity of any affected area, and it is anticipated that these challenges will be exacerbated by the projected impact of climate change. The effects of water-insufficiency stresses have been studied extensively; they limit water and micronutrient uptake and lead to closure of stomata, decline in carbon metabolism, stunted growth, ion/salt toxicity and reduced yield [3, 4].
For plants to survive under such conditions, they must sense and respond to these abiotic stresses rapidly and in a complex manner , through signalling and regulatory pathways [3, 4, 6] mediated by abscisic acid  or ethylene , generally resulting in altered expression of transcription factors , and in many cases in increased expression of genes encoding products required for osmoregulation, cell protection and/or acclimation [10–15]. These modifications may lead to changes in signal transduction, ionic homeostasis, scavenging of reactive oxygen species, accumulation of compatible solutes and growth regulation [3, 6, 16–18]. A common strategy for the identification of overall changes in gene expression under salt stress is to compare the transcriptomes of the targeted species or cultivars using microarrays and/or RNA-Seq technologies . A plethora of comparisons between salt-sensitive and salt-tolerant cultivars of model and non-model plant species, including Arabidopsis[20–22], rice , poplar [24–27], tomato , potato , Medicago truncatula, sugarcane  and olive , have been reported to date. These studies have identified more than 30 families of transcription factors and numerous enzyme-encoding genes involved in responses to salt stress [33, 34]. However, overall changes in gene expression and physiological responses to salt stress vary greatly between different species, particularly between sensitive and non-sensitive pairs of related species [35–39]. It is often difficult to ascertain whether these differences were caused by divergence during the course of evolution or were brought about through adaptive differentiation. It is therefore of interest to compare the overall changes in gene expression that occur in sister species under salt stress, as this will minimise phylogenetic effects.
Here we examine differences in the transcriptomes of two sister desert poplar species under salt stress. Populus serves as a model for elucidating physiological and molecular mechanisms of stress tolerance in tree species [40–42]. Both P. euphratica and P. pruinosa grow in dry deserts with high summer temperatures [43–46]. Both species can tolerate high salinity and survive NaCl concentrations of more than 300 mM  in nutrient solution, and P. euphratica has been used as a model species for studying abiotic responses to salt or drought stress [27, 48–50]. In addition to differences in leaf and hair morphology between the two species, they also occur in different types of habitat. P. euphratica is found in dry deserts with deep underground water while P. pruinosa is distributed in deserts where the underground water is closer to the surface, and therefore more accessible, but also saltier near ancient or extant rivers. It is likely that these two species have diverged due to ecological differentiation, in spite of ongoing gene flow .
In order to test whether regulatory and metabolic pathways in these two species have diverged during their adaptive interactions with salt and other stresses, the transcriptomes of callus subjected to 24 h of salt stress, and control callus samples, from P. euphratica and P. pruinosa were compared in order to identify differentially expressed genes (DEGs) and alternative splicing (AS) events that occurred in response to salt stress. Our results revealed that these two poplar species have both common and species-specific patterns of gene expression under salt stress. The dynamic transcriptome expression profiles of these sister species under salt stress obtained in this study may provide useful insights to inform further analyses of the mechanism of high salinity tolerance in plants. In addition, the genes found to be differentially expressed under salt stress in both species may facilitate the identification of key genes as potentially suitable targets for biotechnological manipulation with the aim of improving poplar salt tolerance.
Summary of the Illumina-Solexa sequencing tags and their matches in the P. trichocarpa genome
Matched genes (%)
Unaligned tags (%)
Total clean tags
Gene ontology (GO) enrichment analyses for salt-responsive genes compared between P. euphratica and P. pruinosa 1
Response to stress
Response to stimulus
Response to biotic stimulus
Amine metabolic process
Cellular polysaccharide metabolic process
Glucan metabolic process
Cellular glucan metabolic process
Cellular macromolecule metabolic process
Peptidase inhibitor activity
Endopeptidase inhibitor activity
Enzyme inhibitor activity
Enzyme regulator activity
Serine hydrolase activity
Serine-type peptidase activity
Transferase activity, transferring hexosyl groups
Xyloglucan:xyloglucosyl transferase activity
External encapsulating structure
On the basis of annotations in the database of Arabidopsis thaliana transporter proteins (http://www.membranetransport.org/all_type_btab.php?oOID=atha1), a total of 99 genes differentially regulated according to all metrics in either P. euphratica or P. pruinosa during salt stress were categorized as transporters (Additional file 4). Among these, 49 DEGs were identified in P. euphratica, of which 25 were up-regulated and 24 down-regulated during salt stress, while 66 were differentially regulated in P. pruinosa, consisting of 51 up-regulated and 14 down-regulated DEGs, and 16 were co-regulated in the two species. For example, we found that POPTR_0003s13470.1, POPTR_0008s14670.1 and POPTR_0006s11590.1, which are homologous to Arabidopsis thaliana Na+/H+ antiporter 18 (AT5G41610, CHX18), potassium transporter 6 (AT1G70300, KUP6) and ABC transporter (AT1G66950, ABCG39), respectively, were co-up-regulated in both species. However, POPTR_0005s04660.1 and POPTR_0014s12700.1, which are homologous to Arabidopsis thaliana sodium/hydrogen exchanger 2 (AT3G05030, NHX2) and potassium transporter 5 (AT4G13420, HAK5) genes, were up-regulated only in P. euphratica. In contrast, POPTR_0004s23680.1 and POPTR_0013s08110.1, which are homologous to Arabidopsis thaliana chloride channel protein CLC-c (AT5G49890, CLC-C) and potassium transporter 2 (AT2G40540, KT2) genes respectively, were up-regulated exclusively in P. pruinosa. These results corroborate previous findings [27, 53–55], and confirm that genes encoding proteins such as sodium and potassium ion transmembrane transporters, and chloride channel and ABC transporters, which are important for maintaining and re-establishing homeostasis in the cytoplasm, are induced to high levels in response to salinity stress .
Transcription factors differentially expressed in the two species under salinity stress
Transcription factor family
List of co-up-regulated DEGs in the two species under salinity stress
malate dehydrogenase (NADP+)
phosphoenolpyruvate carboxykinase [ATP]
ABC transporter G family member 39
alpha/beta-hydrolase domain-containing protein
UDP-glucosyl transferase 73D1
ethylene-responsive transcription factor ERF112
ethylene-responsive transcription factor 1B
Potassium transporter 6
homocysteine S-methyltransferase 3
putative WRKY transcription factor 75
sugar transport protein 13
osmotin-like protein OSM34
glycosyltransferase family protein 2
arogenate dehydratase 6
Phosphorylase-like protein protein
In order to test the consistency of DEGs across different treatments and approaches, we compared DEGs between our results and other available transcriptome studies of the salt stressed poplars. Ottow et al.  examined changes in transcript levels of various genes known to be involved in salt or general stress signaling or adaptation in P. euphratica leaves by dot-blot expression. They identified nine genes with significant changes in response to salt stress. Some of them were be confirmed in the present study, for example, galactinol synthase 2 (GolS2, POPTR_0013s00730.1), calcineurin B-like protein 4 (CBL 4, POPTR_0015s01550.1), alternative oxidase 1A (POPTR_0012s01630.1) and 1-aminocyclopropane-1-carboxylate oxidase (POPTR_0011s00970.1) (Additional file 1). Galactinol synthase (GolS) catalyzes the first step in the biosynthetic pathway of raffinose oligosaccharides using galactose and myo-inositol as substrates and this gene was also up-regulated in plants under cold, heat, drought, and salt stress [21, 61, 62]. Significant increases in galactinol synthase and alternative oxidase after salt stress point to shifts in carbohydrate metabolism and suppression of reactive oxygen species in mitochondria under salt stress . In addition, Gu et al.  identified 54 genes with altered transcript accumulation in the salt-stressed P. euphratica by microarray hybridization. The genes of them, responsible for hydroxyproline-rich glycoprotein, carbonic anhydrase 2, cytochrome P450, aquaporin, sucrose synthase and aspartate aminotransferase were confirmed in these present study. These genes were also revealed to be salt-responsive in other studies [26, 27, 64].
The drought responses of plants are similar to those in response to salinity because both stresses lead to physiological water deficit . Bogeat-Triboulot et al.  provided a comprehensive analysis of P. euphratica subjected to gradual soil water depletion, and observed 110 regulatory and protective genes involved in long-term response to drought. Similar results were also found by Cohen et al.  and Tang et al. . Among them, those genes involved in metabolites of proline, raffinose, galactose, inositol and sucrose under drought stress were found to have changed their expressions in response to salt stress in the presnet study. An increase in galactinol, raffinose and stachyose content may have improved osmoprotection and ROS scavenging when poplars were stressed by drought or salt. However, in the present study, we identified numerous more transcripts with significant up-regulations in both poplars when stressed, including UDP-glycosyltransferase-like protein, FAD-binding and BBE domain-containing protein, putative nucleoredoxin 1, and glyceraldehyde-3-phosphate dehydrogenase. All these newly identified genes should have also played an important role during salt adaptation of two species. Their functions and molecular mechanisms need further clarifications in the future.
Alternative splicing events in response to salt stress in P. euphratica and P. pruinosa
Type of event
Alternative 5’ splice site (A5SS)
Alternative 3’ splice site (A3SS)
Alternative first exon (AFE)
Alternative last exon (ALE)
Total AS events
Loci having AS events
Our transcriptional profiling analysis revealed numerous genes that were differentially expressed in both P. euphratica and P. pruinosa under salt stress. The differential expressions of the selected genes inferred from RNA-seq were confirmed by qRT-PCR data. Gene ontology analyses of these DEGs suggested that GO enrichment in P. euphratica was significantly different from that in P. pruinosa. We found that numerous genes involved in hormone biosynthesis, or encoding transporters or transcription factors, showed different expression patterns between these two species under salt stress. These differences suggest that these two desert poplars may have developed species-specific pathways for adaptation to salinity during the course of ecological speciation in their different salty desert habitats. The results of our comparative analyses imply that different species, even sister species, may employ different genetic pathways to cope with salt stress. This suggests that it may be more difficult than previously anticipated to design salt-tolerant plant cultivars [69, 70]. In order to develop cultivars with high salt tolerance, particular attention should be paid to those genes that are differentially expressed in two or more different species under salt stress. Such genes can be used to facilitate genetic improvement of crops, including cultivated poplars, for growth on saline soils.
Paired-end RNA-seq reads for control callus and salt-stressed callus of P. euphratica and P. pruinosa, which were obtained by Qiu et al.  and Zhang et al. , respectively, were downloaded from the NCBI sequence read archive (accession numbers SRX025571, SRX025568, SRX245887 and SRX245885).
We cultured P. euphratica and P. pruinosa calli induced from the shoot under the same conditions. We then replaced the growth medium for one set with the fresh medium and the same medium but supplemented with 100 mM NaCl (salt stress) for another set. We harvested both sets of calli 24 h later. The calli from P. euphratica and P. pruinosa had the same subculture generation and time and they were highly comparable in terms of physiological state. After RNA extraction and quality determination, we constructed the paired-end cDNA libraries with insert sizes of 200 base pair (bp), and then sequenced the cDNA using an Illumina (San Diego, CA, USA) Genome Analyzer platform according to the manufacturer’s protocols with a read length of 75 bp in two lanes. Image output data from the sequencer was transformed into raw sequence data by base calling.
Raw reads generated by Illumina Genome Analyzer were initially processed to obtain clean reads. We first cleaned raw sequence reads by removing exact duplicates from both sequencing directions. We further cleaned reads by removing adapter sequences as well as reads with too many (>8) unknown base calls (N), low complexity, and low-quality bases (>50% of the bases with a quality score ≤5). Cleaned reads from each library were used for later differential expression analysis in this study.
To determine the level of gene expression, Bowtie2  was used to align RNA-seq reads from the control and salt-stressed samples to transcript sequences from Populus trichocarpa Torr. & A. Gray , using annotation files downloaded from http://www.phytozome.net/poplar (JGI Populus trichocarpa v2.2). No more than a 1 bp mismatch was allowed when taking into account differences between the two species. Reads that mapped to reference sequences from multiple genes were filtered out. The remaining clean reads, which were considered to be distinct, were used for further analysis. Transcript abundances were calculated using eXpress , which outputs read counts and the number of fragments per kilobase of exon per million fragments mapped (FPKM) . Transcripts with FPKM values < 1 in both libraries were filtered out and not subjected to further analysis.
To identify differentially expressed genes (DEGs) in control callus and salt-stressed callus from P. euphratica and P. pruinosa, we applied four independent, widely used tools: Cuffdiff , DESeq , edgeR , and EBSeq . Cuffdiff takes a nonparametric, annotation-guided approach to estimating the means and variances of transcript FPKM values under different conditions, using Student’s t-tests to identify differentially expressed transcripts . In contrast, DESeq, edgeR and EBSeq estimate the means and variances of raw read counts under a negative binomial distribution and use exact tests to identify differentially expressed transcripts. The main difference between DESeq, edgeR and EBSeq is that they use different statistical approaches to estimate variance [74–76]. After the p-values for each expressed genes were obtained by the four tools, the false discovery rate (FDR) was used to justify the p-value by the function p.adjust in R. Sequences were deemed to be differentially expressed if log2(FPKMsalt/FPKMcontrol) > 1 or < -1, and the adjusted p-value (FDR) was < 0.05 as identified by all four metrics.
Gene Ontology (GO) terms were assigned to the identified genes by the blast2GO pipeline  using NCBI databases, followed by functional classification using the WEGO software package . For the comparative analysis of DEGs between P. euphratica and P. pruinosa in response to salinity, singular enrichment analysis (SEA) and parametric analysis of gene set enrichment (PAGE) were performed using the agriGO program (http://bioinfo.cau.edu.cn/agriGO)  with the default parameters, using the P. trichocarpa gene models as background, followed by multiple testing with Bonferroni correction (corrected P-value < 0.05). PermutMatrix (Version 1.9.3; http://www.lirmm.fr/~caraux/PermutMatrix/index.html) was used to cluster genes related to plant hormone biosynthesis according to their mean normalized intensity values .
In order to validate the reliability of RNA-Seq experiments, a total of 21 candidate DEGs highly related to salt stress were selected for qRT-PCR test. These genes were chosen for the qRT-PCR analysis based on two criteria: (i) gene’s expression patterns between these two species under salt stress should be similar; (ii) it should have only one BLAST hit when searching against genes of Arabidopsis thaliana to exclude paralogs. A total of 0.5 μg of DNase I-treated total RNA was converted into single-stranded cDNA using a Prime-Script 1st Strand cDNA Synthesis Kit (TaKaRa, Dalian, China). The cDNA templates were then diluted 20-fold before use. The quantitative reaction was performed on a CFX96 Real-Time PCR Detection System (Bio-Rad, Singapore) using SYBR Premix Ex Taq™ (TaKaRa, Dalian, China). PCR amplification was performed under the following conditions: 30 s at 95°C, followed by 40 cycles of 95°C for 15 s, 60°C for 30 s and then 72°C for 20 s. All primers were designed using PRIMER3 software and were listed in Additional file 8. Three biological replicates based the calli cultured from different individuals with the same subculture and physiological state were performed in order to exclude sampling errors. The relative expression levels of the selected DEGs normalized to an internal reference gene actin was calculated using 2-ΔΔCt method .
We prepared a database of all possible splice junctions between annotated exons in each selected gene and identified new possible junctions using TopHat . We combined these two databases, removing any redundancy between them, and then extracted junction sequences of width 65 bases on each side from all the above junctions. To evaluate which of these junctions were validated by our Illumina reads, we aligned reads from each library separately against the junction sequences, allowing up to one mismatch (in a read of 75 bp). If at least two reads aligned to a splice junction, we considered it to be validated.
Six different types of alternative mRNA processing events were analysed . We first considered skipped exons (SE), in which one or more exons are spliced out of the mature message, and retained introns (RI), in which one or more introns are included in the message. Also included were alternative 5’ splice site (A5SS) and alternative 3’ splice site (A3SS) events, which are particularly difficult to interrogate by microarray analysis because the variably included region is often quite small. Finally, alternative last exons (ALEs) in which alternative use of a pair of polyadenylation sites results in distinct terminal exons, and alternative first exons (AFEs), where alternative promoter use results in mRNA isoforms with distinct 5’ UTRs, were considered.
Digital gene expression
Kyoto Encyclopedia of Genes and Genomes
Number of fragments per kilobase of exon per million fragments mapped
Differentially expressed genes
Parametric analysis of gene set enrichment
Singular enrichment analysis
False discovery rate
Quantitative real-time polymerase chain reaction.
Financial support was provided by the National High-Tech Research and Development Program of China (863 Program, No. 2013AA102605), the National Key Project for Basic Research (2012CB114504), the National Science foundation of China (30972336, 31270652), and the Xinjiang Production & Construction Corps Key Laboratory of Protection and Utilization of Biological Resources in Tarim Basin opening topic fund (BRZD1204). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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