Tree Genetics & Genomes (2017) 13:97 DOI 10.1007/s11295-017-1179-z

ORIGINAL ARTICLE

Transcriptome-wide profiling and expression analysis of two accessions of Paulownia australis under salt stress Yanpeng Dong 1,2 & Guoqiang Fan 1,2 & Zhenli Zhao 1,2 & Enkai Xu 1,2 & Minjie Deng 1,2 & Limin Wang 1,2 & Suyan Niu 1,2

Received: 9 February 2017 / Revised: 7 August 2017 / Accepted: 10 August 2017 # Springer-Verlag GmbH Germany 2017

Abstract Paulownia australis has important economic and ecological values. In this study, the morphological and physiological changes of the leaves in diploid and autotetraploid P. australis under salt stress were analyzed. To detect related genes and gain a comprehensive perspective on the molecular mechanisms underlying salt tolerance in P. australis, transcriptome-wide gene expression profiling was conducted in the leaves of the diploid and autotetraploid P. australis under control and salinity conditions, respectively. Evaluation of the responses against salinity stress revealed the superiority of autotetraploid over diploid in terms of salinity tolerance. Changes in physiological parameters in diploid P. australis (PA2) and tetraploid P. australis (PA4) plants in response to salt stress were measured. Transcriptome data revealed that many of the common unigenes which were involved in accumulation of compatible solutes, oxidative stress detoxification, ion homeostasis, and signal transduction showed significant differences between the two accessions in response to salt stress. A number of salt-responsive unigenes were identified in two accessions of P. australis under salt stress. Furthermore, the Communicated by W.-W. Guo Electronic supplementary material The online version of this article (doi:10.1007/s11295-017-1179-z) contains supplementary material, which is available to authorized users. * Guoqiang Fan [email protected]

1

Institute of Paulownia, Henan Agricultural University, 95 Wenhua Road, Jinshui Area, Zhengzhou 450002, Henan, People’s Republic of China

2

College of Forestry, Henan Agricultural University, 95 Wenhua Road, Jinshui Area, Zhengzhou 450002, Henan, People’s Republic of China

differentially expressed unigenes found to be common in both accessions may be useful genetic resources for further genetic improvement of Paulownia using transgenic approaches. Keywords Paulownia australis . Salt stress . Diploid . Autotetraploid . Transcriptome . RNA-sequencing

Background Plants are constantly exposed to a wide range of abiotic stresses such as high salt, drought, heat, and extremes of temperature. Salinity is one of the most common environmental stresses that severely influence plant growth, development, and productivity (Allakhverdiev et al. 2000). Over the past few decades, there has been a dramatic increase in the scale of salinization of cultivated land, and nearly 20% of the global irrigated agricultural land is affected mostly because of extreme climate change and the high demand of water from other non-agriculture sectors (Munns and Tester 2008). Plants have evolved molecular, cellular, physiological, and metabolic mechanisms to adapt to salt stress. Several mechanisms have been adapted in this process: osmotic adjustment (tolerance against osmotic stress), ion homeostasis (exclusion of Na+ or Cl− from sensitive tissues and compartmentalization of Na+ or Cl− in specific cells, cellular organelles, or tissues), and scavenging of toxic compounds (Munns and Tester 2008). Extensive molecular and biochemical studies of the mechanisms underlying salt-stress responses in model plants have shown that the responses to salt often involve complex gene regulation events (Bohnert et al. 1995), such as intracellular signaling and regulatory pathways (Munns and Tester 2008) mediated by plant hormones (Ma et al. 2009), induction of specific transcription factors (Urano et al. 2010), and/or increased expression of functional genes that encode products

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required for cell protection, osmoregulation, and/or acclimation (Forrest and Bhave 2007; Meiri and Breiman 2009; Tyerman et al. 2002). For example, in Arabidopsis thaliana, the signal transduction components of the salt overly sensitive pathway that mediates ion homeostasis and contributes to salt tolerance have been identified (Cheng et al. 2004; Gong et al. 2001; Liu et al. 2000). The activation of a mitogen-activated protein kinase (MAPK) signaling cascade could mediate salt stress tolerance in plants (Teige et al. 2004). Previous studies have revealed that calcium-dependent protein kinases play crucial roles in Ca2+-mediated signal transduction in plants’ response to salinity stresses (Romeis et al. 2001). Some studies have focused on genes related to osmolyte synthesis and detoxification in plants under salt stress (Hong et al. 2000; Nanjo et al. 1999). These stress responses may contribute to changes in ionic homeostasis, signal transduction, accumulation of compatible solutes, and scavenging of reactive oxygen species (ROS) (Hasegawa et al. 2000). Paulownia australis is a fast-growing and deciduous hardwood species that is native to China where it has been cultivated for over 2000 years. It has also been introduced to many other countries and can now be found throughout the world, except Antarctica. This species has important economic and ecological values, including being used as high-quality hardwood timber, in ornamental/garden applications, and for land reclamation (Wang et al. 2010). P. australis is naturally distributed in areas where the soil is salinized and has excellent tolerance to salt stress. Therefore, it is an ideal species to study the molecular mechanisms of salt tolerance in plants. Polyploid plants exhibit many improved properties, for example, higher stress resistance compared with their diploid relatives (Chao et al. 2013; Tan et al. 2015). To investigate the possible molecular mechanisms of salt stress resistance of polyploidy, autotetraploid P. australis was obtained from the leaves of diploid P. australis using colchicine treatment, and an in vitro plantlet regeneration system has been established. The autotetraploid plants were generally superior in several characteristics and exhibited higher tolerance to biotic and abiotic stress than their diploid relatives (Fan et al. 2009; Xu et al. 2015). In recent years, our group has applied transcriptome comparative analysis to study the molecular responses of diploid and autotetraploid Paulownia plants to drought stress through identifying genes that might be involved in response to water deficit (Dong et al. 2014; Xu et al. 2015). As a result, many drought-related genes and regulatory factors have been identified at the genome-wide level, and differences in the transcriptome remodeling between diploids and autotetraploids under drought stress have also been reported. However, similar information for the responses of diploid and autotetraploid Paulownia to salt stress is largely unknown. Different from the previous studies, the novel compared accessions and the novel backgrounds of sequencing and assembling were emerged.

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The major goal of the present study was to gain insights into the complex molecular mechanisms of P. australis plants’ response to salt stress and to investigate whether regulators and metabolic pathways in diploids and autotetraploids differed in salt-stressed conditions. To this end, the transcriptomes of the leaves of diploid and autotetraploid accessions of P. australis (controls and salt-treated seedlings) were sequenced and analyzed. Our findings may provide a useful genetic resource for further analyses on the molecular mechanisms of the salinity tolerance in this species. Furthermore, the unigenes differentially expressed under salt stress in the diploid and autotetraploid plants may be suitable candidate targets for biotechnological manipulation with the aim of improving salt tolerance in Paulownia plant species.

Materials and methods Plant material All the plant materials used in this study were obtained from the Institute of Paulownia, Henan Agricultural University, Zhengzhou, Henan Province, China. Uniformly grown tissue cultured seedlings of diploid P. australis (PA2) and tetraploid P. australis (PA4) were cultured in 100-mL triangular flasks on 1/2 Murashige and Skoog (MS) medium for 30 days before being clipped from the roots. Samples with the same height and crown size were then transferred into plastic pots (30 cm in diameter at the bottom and 20 cm deep) containing the same amount of ordinary garden soil, one pot for each plant. Prior to salt stress treatment, the plants were housed randomly in an outdoor nursery for 50 days. Twelve plants from each accession were selected, including three controls and nine plants for salt treatment (divided into three groups). For salt treatment, the PA2 and PA4 seedlings in the three groups were treated with 0.2, 0.4, or 0.6% NaCl solution (1 L) every 2 days for 15 days. The PA2 and PA4 seedlings in the control group were irrigated with tap water every 2 days for 15 days. Healthy leaves (second leaf from the apex) were harvested at 15 days, and equal numbers of leaves from each plant in each of the replicate groups were pooled to form the four samples. The leaf tissues were frozen in liquid nitrogen and then stored at − 80 °C for RNA isolation and further analysis. Physiological measurements Physiological measurements were made on single, fully expanded leaves (third and fourth leaves from the terminal bud of a twig) that were picked from the control and salttreated samples between 8:30 and 11:00 am on day 15. The measurements were made on three replicates of the leaves from the same plants.

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The relative water content (RWC) of the leaf was calculated as (fresh weight − dry weight) / (turgid weight − dry weight) × 100 (Barrs and Weatherley 1962). Chlorophyll content was measured in the described method previously (Bojović and Stojanović 2005) and calculated as described by Arnon (1949). Malondialdehyde concentration was measured using the thiobarbituric acid method as described by Hodges et al. (1999). Relative electrical conductivity (REC) was measured as described previously (Liu et al. 1996). Superoxide dismutase activity was determined using the nitroblue tetrazolium method reported by Dhindsa et al. (1981). Values were expressed in units/gmHb (Sun et al. 1988). Total soluble protein content was measured following the procedure of Guy et al. (1992), and protein concentration was measured as described by Bradford (1976) using bovine serum albumin as the standard. Proline content was measured using the acid ninhydrin method of Bates et al. (1973). Soluble sugar content was measured using the anthrone colorimetry method described by Irigoyen et al. (1992).

Statistical analysis Analyses of variance (ANOVA) of the parameters were performed using the MSTAT-C software package (https:// msu.edu/~freed/mstatc.htm) and the SPSS program (http:// www.spss.com.cn/). The mean values of each of the measured traits were compared using the least significant difference test at a 5% level of probability according to Armitage et al. (2008).

RNA extraction and double-stranded cDNA synthesis The PA2 and PA4 seedlings from the 0.4% NaCl-treated group were chosen for sequencing based on the physiological measurements (See the BResults^ section for details). Total RNA was extracted from the leaves of the four samples treated with either 0.4% NaCl (PA2T and PA4T) or water (PA2W and PA4W) using TRIzol reagent (No. 155960189, Invitrogen, Carlsbad, CA, USA). RNAs with a poly (A) tail were purified from the total RNA using oligo (dT) magnetic beads. Double-strand cDNA was synthesized using SuperScript™ II reverse transcriptase (No. 18064014, Invitrogen, Carlsbad, CA, USA) followed by DNA polymerase I (No. 18162016, Thermo Scientific, Waltham, MA, USA) and RNase H (No. 18021071, Invitrogen). The PCR products of the cDNA were qualified using a 2100 Bioanalyzer (Agilent, Palo Alto, CA, USA) and quantified using an ABI StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Finally, the cDNA libraries were sequenced on an Illumina Genome Analyzer IIx platform (Illumina, San Diego, CA, USA) by Beijing Genomics Institute, Shenzhen, China.

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RNA-Seq data processing, de novo assembly, and annotation The raw image data were transformed by base calling into sequence data (raw reads), which were stored in FASTQ format. Processing of the raw reads and filtering out dirty raw reads, such as reads with adaptors, unknown nucleotides larger than 5%, and low-quality reads which the percentage of low-quality bases (base quality ≤ 10) is more than 20%, were performed using Dynamic Trim in the Solexa QA package (Illumina). The Trinity software (kmer = 25) program (Grabherr et al. 2011) was used for transcriptome de novo assembly of the remaining (clean) reads. The assembled sequences can be taken into further process of sequence splicing, and redundancy removing with sequence clustering softwares, which could not be extended further on either end, was defined as unigenes (Supplementary material 1). The ESTScan program (Iseli et al. 1999) was used to detect open reading frames (putative protein coding regions) in the unigene sequences. Because of lack of genome and EST information of Paulownia species, BLASTX (https://blast.ncbi.nlm.nih.gov/ B l a s t . c g i ? P R O G R A M = b l a s t x & PA G E _ T Y P E = BlastSearch&LINK_LOC=blast) alignment (evalue < 0. 00001) between unigenes and protein databases like NCBI non-redundant protein sequence database (Nr) (ftp://ftp.ncbi. nih.gov/blast/db/nr), Swiss-Prot (http://www.ebi.ac.uk/ swissprot/), Eukaryotic Orthologous Groups (KOG) (http:// www.ncbi.nlm.nih.gov/COG/grace/shokog.cgi), and Kyoto Encyclopedia of Genes and Genomes (KEGG) (http://www. genome.jp/kegg/) were performed, and the best aligning results were used to decide sequence directions of unigenes. If results of different databases conflict with each other, a priority order of NR, Swiss-Prot, KEGG, and COG should be followed when deciding sequence direction of unigenes. When a unigene happens to be unaligned to none of the above databases, a software named ESTScan (http://estscan. sourceforge.net/) will be introduced to decide its sequence direction. Complete unigene transcripts that aligned to the KOG database were assigned possible functions. The Blast2GO program (Conesa et al. 2005) was used to analyze the gene ontology annotations assigned to the unigenes based on the Nr annotations of the best BLASTX hits. Then, the WEGO software (Ye et al. 2006) was used to perform a GO functional classification for the unigenes. The annotated sequences were assigned to biological pathways based on searches against the KEGG database (Kanehisa et al. 2008). Gene expression quantification and differential expression analysis The software of RSEM (Li and Dewey 2011) was selected to quantify gene and isoform abundances from paired-end RNASeq data. The value of kmer we used in the assembly process

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was 25. We obtained the read counts in every unigene. Then the trimmed mean of M (TMM) was used to normalize the read counts into fragments per kb per million reads (FPKM) values. The FPKM values were convenient for comparing the expression level of unigenes in four libraries. Differentially expressed unigenes (DEUs) among the four samples were identified using edgeR (version 3.8.5) (Robinson et al. 2010) using the read counts that were calculated by RSEM. The edgeR owned internal homogenization which tested for differential expression using the exact test method. P values reported by edgeR were used to calculate false discovery rates (FDR) for each unigenes. A FDR and absolute value of the |log2Ratio| > 1 were used as the threshold to judge the significance of the differences in gene expression (Benjamini and Yekutieli 2001). GO terms enriched in the DEUs and KEGG pathways that were related to salt stress were identified using the Plant MetGenMAP system (Joung et al. 2009). Correlation coefficients of the expression of the duplicate samples The raw data using duplicate well-watered diploid P. australis were screened out as mentioned above. The generated clean data were then compared to the original project of the assembled all-unigenes. The expression of the unigenes was calculated using FPKM methods (Mortazavi et al. 2008). In order to test the repeatability of our data from RNA-Seq, the correlation coefficients of logarithmic values of the expression of the duplicated samples were then calculated. Quantitative real-time PCR analysis Fifteen of the salinity responsive unigenes identified in the transcriptome sequencing data were selected for validation by qRT-PCR using StepOne-Plus real-time PCR detection system (No. 4376373, Thermo Scientific). Total RNA from the leaves of the control and salinity-stressed PA2 and PA4 samples were extracted using TRIzol (No. 155960189, Invitrogen). The first-strand cDNA was synthesized from the total RNA using the PrimeScript RT reagent kit (No. RR037A Takara, Dalian, China) according to the manufacturer’s instructions. Specific primers were designed using Beacon Designer (version 7.7) (Premier Biosoft International, Ltd., Palo Alto, CA, USA) and are listed in Supplementary material 2. The cDNA was amplified in a CFX96TM Real-Time System (Bio-Rad) with Sso Fast Eva Green Supermix (BioRad) according to the manufacturer’s instructions. The PCR reaction cycles were as follows: 95 °C for 1 min, followed by 40 cycles of 95 °C for 10 s and 55 °C for 15 s. Relative expression levels of the selected unigenes were calculated using the 2−ΔΔCt method (Livak and Schmittgen 2001) and normalized with 18S rRNA from Paulownia fortunei.

Results Morphological changes of P. australis during salt treatment Evaluation of the responses of PA2 and PA4 against salinity stress revealed the superiority of PA4 over PA2 in terms of salinity tolerance. The phenotypic and physiological responses of PA2 and PA4 to salt stress were assessed using seedlings exposed to 0.4% NaCl for 15 days. There were no significant differences in stress phenotype between PA2 and PA4 under the control conditions (Fig. 1a and e). Overtime, symptoms of dehydration gradually worsened in the NaCltreated PA2 and PA4 plants. The leaves of the PA2 seedlings drooped after 5 days of salt stress, whereas the leaves of the PA4 seedlings remained turgid and bright green (Fig. 1b and f). After 10 days, the stems of the PA2 seedlings began to bend and the leaves were paler and more wilted than the leaves of the PA4 plants (Fig. 1c and g). After 15 days, the PA2 and PA4 plants both showed stunted growth under salt stress; however, the PA4 plants showed less wilting and much better development than the PA2 plants (Fig. 1d and h). Changes in physiological parameters in PA2 and PA4 plants in response to salt stress The RWC and chlorophyll content of the leaves from the PA2 and PA4 accessions declined after being treated by salt, as characterized by their significantly lower RWC and chlorophyll content compared with the controls. However, the RWC and chlorophyll content were always higher in PA4 than in PA2 in both the salttreated and control plants (Fig. 2a and b). On exposure to salt stress, REC and malondialdehyde (MDA) content showed overall increased tendencies in both the PA2 and PA4 plants; however, the REC and MDA levels in the PA2 plants were much higher than in the PA4 plants over the entire 15-day treatment process (Fig. 2c and d). Superoxide dismutase (SOD) activity and soluble protein content initially increased under the 0.2 and 0.4% NaCl treatments in both accessions and decreased dramatically under the 0.6% NaCl solution. In particular, PA2 showed lower SOD activity and soluble protein content compared with PA4 at the end of the 15-day experiment (Fig. 2e and f). Additionally, the soluble sugar and proline contents in PA2 and PA4 tended to increase as the salt treatment strengthened (Fig. 2g and h). The NaCl-treated PA4 plants accumulated higher levels of soluble sugar and proline in response to the three different NaCl concentration treatments than the NaCl-treated PA2 plants. Transcriptome sequencing and de novo assembly of the P. australis reads Overflow of the de novo transcriptome assembly of the RNASeq data was shown in Supplementary material 1. Over 256

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Fig. 1 Phenotype change of diploid and autotetraploid P. australis under salinity stress (0.4% NaCl solution). a PA2W, well-watered diploid. b PA2T-1, 5-day salt-treated diploid. c PA2T-2, 10-day salt-treated

diploid. d PA2T-3, 15-day salt-treated diploid. e PA4W, well-watered tetraploid. f PA4T-1, 5-day salt-treated tetraploid. g PA4T-2, 10-day salt-treated tetraploid. h PA4T-3, 15-day salt-treated tetraploid

million raw reads were generated from the four libraries. After removing low-quality reads, 44.6 million (PA2W), 73.8 million (PA2T), 47.2 million (PA4W), and 75.3 million (PA4T) clean reads with an average GC content of 44% remained. The Q20 (sequencing error rate, 1%) for the four libraries were 83.1, 86.7, 88.5, and 91.3% for PA2W, PA2T, PA4W, and PA4T, respectively. The clean reads in the four libraries were assembled de novo using the trinity program, finally which generated a total of 171,743 unigenes. The total length of these unigenes was 199,969,905 bp, with a mean length of 1164 bp and an N50 (medium of the length) of 2097 bp. Among these unigenes, 98,812 (57.5%) were longer than 500 bp, and 67,980 (39.6%)

were longer than 1000 bp (Table 1). The length distribution of these unigenes is shown in Supplementary material 3. The correlation coefficient of the expression of PA2 was exhibited in Fig. 3. The duplicates indicated linear correlations with its corresponding one, and the value of Pearson r was 0.9722.

Fig. 2 Effects of salt stress on P. australis physiology. PA2 represents diploid P. australis, PA4 represents autotetraploid P. australis, and 0.2, 0.4, and 0.6% NaCl solution (1 L) were used. a Effect of salt stress on leaf relative water content (RWC). b Effect of salt stress on leaf chlorophyll content. c Effect of salt stress on leaf relative electrical conductivity

Annotation of the unigenes against public databases Similarity searches using the BLASTX program (E value < 1.0E− 5) detected 34,177 (88.63%), 34,606 (89.75%), 27,502 (71.33%), 9562 (24.80%), and 16,309 (42.29%) of the unigenes that had significant matches with sequences in

(REC). d Effect of salt stress on leaf malondialdehyde (MDA) content. e Effect of salt stress on leaf superoxide dismutase (SOD) activities. f Effect of salt stress on leaf soluble protein content. g Effect of salt stress on leaf proline content. h Effect of salt stress on leaf soluble sugar content

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Table 1 Summary of the sequencing and assembly data of the P. australis transcriptome

Statistics of data production Original data Number of raw reads After filtering Number of clean reads Total length (bp) Q20 percentage (%) GC percentage (%) Average length (bp) Assembly Total number of unigenes Total length (bp) Length of N50 (bp) Average length of unigenes (bp) Number of unigene (≥ 500 bp) Number of unigene (≥ 1000 bp)

PA2

PA2T

PA4

PA4T

48,156,310

78,262,386

49,911,984

80,366,542

44,554,742 4,341,985,315 83.1% 44% 97.45

73,841,044 6,156,590,415 86.7% 44% 83.38

47,200,652 4,560,562,512 88.5% 44% 96.62

75,304,356 6,626,125,064 91.3% 44% 87.99

171,743 199,969,905 2097 1164.36

the Nr, KEGG, Swiss-Prot, KOG, and GO databases, respectively (Supplementary material 4). Overall, 27,055 (70.09%) unigenes shared similarities with sequences in at least one of these five public databases. Of the 38,558 annotated unigenes, 7551 found no matches in any of these databases.

98,812 67,980

Functional and metabolic pathway analysis of the unigenes Based on sequence similarity, 9562 unigenes (24.80%) were assigned to 25 different functional categories in the KOG database (Supplementary material 5), and 16,309 unigenes (42.29%) were categorized into 49 functional groups under the three main GO categories: biological process, cellular component, and molecular function (Supplementary material 6). To assign the annotated unigenes to potential metabolic pathways, they were mapped against the Enzyme Commission numbers (EC numbers) in the KEGG database, and 34,606 unigenes (89.75%) were assigned to 317 KEGG pathways (Supplementary material 7). Identification and analysis of potential differentially expressed unigenes

Fig. 3 Correlation coefficients of the expression of duplicate samples. Xaxis represents the logarithmic value of diploid expression, while Y-axis represents the logarithmic value of the corresponding duplicate sample

Four pairwise comparisons of the four transcriptomes were performed to further understand the biology of the salt response and tolerance of PA2 and PA4 plants. In total, 6845 DEUs (3109 up-regulated, 3736 down-regulated) were identified in the PA2T vs. PA2W comparison. 6724 DEUs (3527 up-regulated, 3197 down-regulated) were identified in the PA4T vs. PA4W comparison. 10,383 DEUs (4833 up-regulated, 5550 down-regulated) were identified in the PA4W vs. PA2W comparison. 4407 DEUs (2353 upregulated, 2054 down-regulated) were identified in the PA4T vs. PA2T comparison. To identify candidate genes associated with salt response and tolerance, 2360 co-regulation DEUs

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(similar co-regulation and opposite co-regulation) were selected from two comparisons of the PA2T vs. PA2W and PA4T vs. PA4W (Supplementary material 8). Additionally, 2470 DEUs were selected from the PA4W vs. PA2W and PA4T vs. PA2T comparisons (Supplementary material 9). Among the selected DEUs, 375 DEUs were common in all four transcriptomes (Supplementary material 10). Several unigenes that were demonstrated previously to mediate salinity, drought, and osmotic stress tolerance, such as SOD, AP2/ERF transcription factor, lipoxygenase, and late embryogenesis abundant protein were among the most abundant transcripts. Among these DEUs, some had either no annotated or were annotated with unknown function (including hypothetical and predicted proteins). Some of these unigenes may be found to contribute to salt tolerance when studied further. The results of the unigenes that function in the accumulation of compatible solutes, oxidative stress detoxification, ion homeostasis, and salt stress in the discussion section were shown in Supplementary material 11. The co-regulated DEUs in the four libraries were classified based on their GO annotation (Fig. 4). GO functional enrichment analyses were performed to predict the biological functions of the selected DEUs using an FDR corrected P ≤ 0.05 as the threshold. As expected, stress-responsive processes such as response to water deprivation, response to abiotic stimulus, cellular response to salt stress, response to metal ion, and

Fig. 4 GO function analysis results for the commonly differentially expressed genes in four libraries. PA4T, 15-day salttreated tetraploid. PA4W, wellwatered tetraploid. PA2T, 15-day salt-treated diploid. PA2W, wellwatered diploid

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cellular response to osmotic stress were highly enriched. Signal-related processes such as hormone-mediated signaling pathway, activation of MAPK activity involved in osmosensory signaling pathway, and intracellular signal transduction also were highly enriched among the DEUs. Under transportrelated categories, terms associated with carbohydrate transmembrane transport, calcium ion transport, hormone transport, proline transmembrane transport, ion transmembrane transport, and sodium ion transmembrane transport were highly significant among the DEUs. Interestingly, among protein homeostasis metabolism-related processes, transport, localization, catabolism, and folding were predominant. In addition, some metabolic terms such as glutamine metabolic process, inositol metabolic process, trehalose metabolic process, maltose metabolic process, hormone metabolic process, and raffinose metabolic process were overrepresented among the DEUs. The co-regulated DEUs were mapped to terms in the KEGG database and compared against the KEGG terms for all the P. australis unigenes to identify pathways that were significantly enriched (Supplementary material 12). Validation of salinity-responsive DEUs by qRT-PCR To validate the reproducibility and accuracy of the transcriptome RNA-Seq data, 15 unigenes were selected for qRT-PCR analysis. The results showed that although the absolute fold changes of the

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expression levels of the selected unigenes varied between the RNA-Seq data and qRT-PCT data, the trends of the gene expression changes detected by the two different methods were largely consistent (Fig. 5a and b).

Discussion In this study, we exposed two P. australis genotypes (PA2 and PA4) to 0.2, 0.4, or 0.6% NaCl treatment to analyze the differences in the salt tolerance. A comparison of the physiological parameters between the two accessions revealed the difference in salt tolerance between the two accessions and may explain why the PA4 plants could survive better and maintain higher growth than PA2 plants in salt conditions. This result is in agreement with the findings of our previous studies (Zhang et al. 2013). Because of the lack of genome and EST information for Paulownia species, it is perhaps unsurprising that 7551 of the 38,558 annotated unigenes found no matches in any of five public databases, indicating that some of these unigenes may be putative novel sequences specific to Paulownia. We found that the numbers of DEUs in the salt-treated and untreated plants were quite different and a much smaller number of unigenes were induced in PA4 compared with in PA2. These results indicated that the molecular responses to salt stress were strikingly different in PA2 and PA4. A similar finding was reported previously in rice where the salt-sensitive IR29 had a larger number of genes with altered expression levels in response to salinity stress compared with the salt-tolerant FL478 (Walia et al. 2005). Although the PA2 and PA4 transcriptomes showed distinctive differences in their gene expression profiles in response to salinity, a large number of the unigenes overlapped between the two accessions, which is consistent with the fact that the two accessions were derived from a common genetic background. Many of the up-regulated DEUs in PA2 and PA4 were involved in transports, transcription, signal transduction, and metabolism, but in PA2, their transcript abundances were higher levels than in PA4 under control conditions and then remained relatively stable under salt stress compared with the PA4. This result may imply that PA4 exhibit better growth and better salt-stress tolerance than PA2 plants because of the significantly increased abundance of metabolism- and defense-related unigenes in their transcriptome. Based on the GO and KEGG analyses, the expression patterns of the DEUs involved in osmotic regulation, ROS scavenging system, and Na+ homeostasis or DEUs that have regulatory functions under salinity stress were further analyzed. Unigenes involved in the accumulation of compatible solutes To maintain osmotic balance and cellular structures during salt stress, many plants accumulate compatible osmolytes that can

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mitigate the damage of osmotic stress. Several studies have demonstrated that tolerance to salinity or drought stress was strongly correlated with the accumulation of soluble carbohydrates in plants (Kerepesi and Galiba 2000; Tan et al. 1992). In a previous study, mannitol was found to accumulate as a major biochemical product in higher plants where it played a role as osmoprotectants under osmotic stress (Wang et al. 2005). Two unigenes encoding a mannitol dehydrogenase (mtlD), which is involved in mannitol synthesis, were found to be upregulated in both PA4T and PA2T plants. When mtlD was transformed in wheat, the plant exhibited enhanced tolerance to osmotic stress (Davies et al. 1999). The introduction of mtlD into sorghum was also reported to elevate its droughtand salinity-stress response (Davies et al. 1999). Galactinol and raffinose play important roles in adapting to cold, heat, drought, and salt stresses in plants (Ottow et al. 2005; Panikulangara et al. 2004; Taji et al. 2004). Galactinol synthase (GolS) is a key enzyme that catalyzes the first step in the biosynthetic pathway of raffinose oligosaccharides (Taji et al. 2002). Arabidopsis and Populus euphratica plants that overexpressed GolS showed elevated salt-stress tolerance because of the accumulation of galactinol and raffinose (Ottow et al. 2005; Taji et al. 2004). In transgenic Arabidopsis, overexpression of AtGolS1 and AtGolS2 were also reported to protect plants from oxidative damage (Nishizawa et al. 2008). Significant increases in GolS after salt stress could enhance plants’ osmoprotection as well as ROS scavenging (Nishizawa et al. 2008). These results suggest that the GolS gene family may also be important in the salt response of P. australis. Further, trehalose acts as a stress protectant, reserve sugar, protects membranes against denaturation, and stabilizes proteins (Yoshida et al. 2006). Over-expression of a trehalose-phosphate synthetase (AtTPS) in transgenic Arabidopsis and rice plants has been reported to elevate trehalose, glucose, fructose, and sucrose contents as well as enhance the plants’ tolerance to drought, salt, and osmotic stress (Yoshida et al. 2006). One unigene encoding homolog of TPS, which functions in ABA and osmotic stress signaling (Kolukisaoglu et al. 2004), was significantly up-regulated in PA4 compared with PA2 plants, implying that the product of TPS may be important in regulating salt-stress tolerance in PA4. It has been reported that sucrose can act as a compatible osmolyte and play a critical role in plant salinity tolerance (Ashraf and Harris 2004). In this study, one unigene encoding a member of the sucrose synthase (SUS) family was downregulated in salt-stressed PA4. Its homolog in Arabidopsis (SUS4) was found to be involved in sucrose, glucose, and fructose accumulation (Bieniawska et al. 2007) and, in P. euphratica, SUS4 was reported to be significantly downregulated in response to osmotic stress (Tang et al. 2013), suggesting that the down-regulation of SUS4 might increase the accumulation of soluble carbohydrates in response to salt stress in PA4 plants.

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ƒFig.

5 a qRT-PCR analyses of candidate salt response genes. b Expression level of 15 DEUs in digital gene expression profiling under 0.4% salt treatment. a Comp78915_c0_seq1, calcium-binding protein. b Comp92036_c0_seq1, cationic amino acid transporter 5-like. c Comp85833_c0_seq2, potassium transporter 2-like. d Comp83607_c0_ seq5, chaperonin. e Comp92213_c0_seq4, sugar transporter. f Comp84796_c0_seq3, amino acid transporter. g Comp90878_c0_seq1, electron carrier. h Comp72313_c0_seq1, zinc finger protein. i Comp93829_c0_seq1, arginase 1. j Comp92857_c0_seq1, mannan synthase. (k) Comp86996_c0_seq4, WRKY transcription factor. l Comp90624_c0_seq14, serine/threonine-protein kinase. m Comp84469_c0_seq1, Phosphate transporter. n Comp92213_c0_seq6, Hexose carrier protein HEX6. o Comp85208_c0_seq1, leucine-rich repeat receptor-like protein kinase.18S rRNA was used as the internal reference gene. Standard error of the mean for three technical replicates is represented by the error bars. A, PA2W, well-watered diploid; PA2T-1, 3-day salt-treated diploid; PA2T-2, 7-day salt-treated diploid; PA2T-3, 11day salt-treated diploid; PA2T, 15-day salt-treated diploid; B, PA4W, well-watered tetraploid; PA4T-1, 3-day salt-treated tetraploid; PA4T-2, 7-day salt-treated tetraploid; PA4T-3, 11-day salt-treated tetraploid; PA4T, 15-day salt-treated tetraploid

Glycine betaine and proline are major organic osmolytes that accumulate in plants in response to environmental stresses by stabilizing sub-cellular structures (e.g., proteins and membranes) under stress conditions (Hsu et al. 2003). Recent study has revealed that the accumulation of glycine betaine in rice could mitigate the adverse effects of salt stress (Rahman et al. 2002). In our study, a unigene encoding betaine aldehyde dehydrogenase, which is involved in glycine betaine biosynthesis, was up-regulated in PA4compared with PA2,

Fig. 5 continued.

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suggesting that glycine betaine synthesis may play a role in the response of PA4 to salt stress. Proline functions as an osmolyte for osmotic adjustment to stabilize cell proteins and structures under abiotic stress (Seki et al. 2007). The accumulation of proline in plants in response to salinity stress was reported previously (Huang et al. 2013). We found that salt stress induced the accumulation of proline in both PA2 and PA4 leaves. In plants, the proline biosynthetic pathway starts with the phosphorylation of glutamate or ornithine, and increased proline synthesis was associated with the upregulation of Δ1-pyrroline 5-carboxylate synthetase (P5CS) and P5C reductase (P5CR) and the down-regulation of proline dehydrogenase (ProDH) (Kishor et al. 2005). In the present study, most of the unigenes encoding proteins in the proline biosynthetic pathway were found to be co-up-regulated in both accessions but, under salt stress, the fold changes in PA4 were greater than in PA2, especially for DEUs encoding P5CS and P5CR. Conversely, unigenes encoding ProDH, the key enzyme in the proline degradation pathway, were down-regulated in PA4 and up-regulated in the PA2 in response to salinity, suggesting that these enzymes could be critical in enhancing the osmotic stress tolerance of PA4. These results are consistent with the physiological parameters that showed increasing proline content in PA2 and PA4 plants and increased accumulation of proline in PA4 compared with PA2 under salinity stress. In the present study, many of the DEUs between the two accessions under salt stress were related to metabolites of carbohydrates and amino acids. Similar results were reported in finger millet (Zhang et al. 2014) and poplar (Rahman et al. 2014) in response to salt stress. However, a previous study

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found that, in P. euphratica, the accumulation of soluble carbohydrates and proline did not necessarily contribute to osmotic adjustment under excess salinity (Zhang et al. 2013). This finding may indicate that different mechanisms of osmotic adjustment operate in different plants in response to salinity stress, but this requires further investigation. In addition to proline and soluble carbohydrates, other osmolytes can ameliorate the negative effects of high ionic levels, for example, by preventing the unfolding and precipitation of proteins (Vinocur and Altman 2005; Welch and Brown 1996). In our transcriptome data, we also found several unigenes encoding heat shock proteins, dehydrins, and late embryogenesis abundant (LEA) proteins that were upregulated in PA4 compared with PA2. Unigenes involved in oxidative stress detoxification Biotic and biotic stresses not only cause osmotic and ionic imbalances in the plant cells but also can lead to the formation and accumulation of ROS such as hydrogen peroxide (H2O2), hydroxyl radicals (HO−), and superoxide radicals (O2−). At low levels, these ROS may function as signal molecules, whereas excessive amounts may inhibit photosynthesis as well as cause oxidative damage to normal cell (Apel and Hirt 2004). Accumulation of raffinose, stachyose, proline, and galactinol may enhance ROS scavenging when plants are salt stressed (Miller et al. 2010). Additionally, microarray and transcriptome studies have revealed that plants have developed effective antioxidant systems to minimize oxidative damage, including various antioxidants and ROS-scavenging enzymes such as guaiacol peroxidase (POX), catalase (CAT), SOD, glutathione S-transferase (GST), lactoyl glutathione lyase, ascorbate peroxidase (APX), and glutathione peroxidase (Miller et al. 2010). Our physiological studies showed that the concentrations of proline and soluble sugar increased rapidly in P. australis plants as the salt concentration increased and enhanced SOD activity was also observed. These results indicated that P. australis may have a similar ROS scavenging mechanism as other plant species and this mechanism may improve the ability of PA2 and PA4 plants to reduce oxidative damage. In the present study, numerous unigenes encoding glutathione peroxidase, CAT, SOD, GST, and APX were significantly up-regulated in both accessions and were more specifically up-regulated in PA4 under salinity stress. Interestingly, one unigene (comp66971_c0_seq1) encoding SOD was found to be up-regulated in PA4T and down-regulated in PA2T. Unigenes involved in ion homeostasis and salt stress signaling pathways When plants are subjected to salinity stress, salinity perception occurs by osmotic and ionic stress signaling and regulatory pathways. Signals are perceived by receptors embedded in the

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plant plasma membrane, cytoplasm, or nucleus, and they, together with cell membrane transport pathways (e.g., ion channels and ion carriers), participate in stress signaling pathways (Peng et al. 2014). In our study, a large number of unigenes encoding receptors or transporters were found to be differentially regulated during salinity stress in the PA2 and PA4 plants. Notably, unigenes encoding ABC transporters, potassium transporters (KT2 and KT5), sodium transporter, potassium channel, and aquaporin proteins were co-upregulated in both accessions, but the transcript abundances of these DEUs indicated a higher fold change in PA4 than in PA2. In contrast, two sodium transporters were found to be significantly up-regulated in PA4 but down-regulated in the PA2 in response to salinity. Interestingly, 12 unigenes encoding a cyclic nucleotide-gated ion channel protein, which is involved in the initiation of cytosolic Ca2+-dependent signal transduction, were up-regulated in PA4, whereas four unigenes encoding the same protein were down-regulated in the PA2. After stress signals are perceived by the membrane receptors, a complex intracellular signaling cascade is activated. Salt stress signaling pathways can be classified as follows: (1) Ca2+-dependent SOS pathway regulates ion homeostasis; (2) Ca2+-dependent signaling-activation of LEA-type genes, such as CDPKs; and (3) osmotic/oxidative stress signalingMAPK modules pathway. In plants, the SOS pathway involves ion transporters that are likely to manage excess extracellular or intracellular Na+ under salt stress. The ionic aspect of salt stress is sensed via calcium sensors such as calcineurin B-like (SOS3/CBL4), which interact with CBL-interacting protein kinases (SOS2/CIPK24). The SOS3-SOS2 protein kinase complex directly phosphorylates and activates ion transporters such as SOS1 (Na+/H+ antiporter), resulting in efflux of excess Na+ ions (Batelli et al. 2007; Qiu et al. 2002). A genetic analysis indicated that the SOS3-SOS2 complex also inhibited Na+ transporter HKT1 activity, thus restricting Na+ ions influx into the cytosol. Additionally, this complex was reported to interact with SOS4, which encodes pyridoxal kinase, and SOS5, which encodes AGP-like (arabinogalactan proteins-like) domains, thereby affecting other salt-mediated pathways and leading to ionic homeostasis (Mahajan et al. 2008). The signal transduction components of the SOS cascade have been clearly revealed in A. thaliana (Gong et al. 2001), poplar (Tang et al. 2010), chickpea (Molina et al. 2011), and Suaeda salsa (Li et al. 2011). In our study, several unigenes involved in the SOS pathway were found to be differentially regulated in both accessions under salinity stress. Three unigenes encoding SOS2 were upregulated in PA4 under salinity and down-regulated in PA2. In addition, unigenes encoding Na+/H+ antiporter (SOS1) and a calcium-binding protein (SOS3) were found to be up-regulated in both accessions under salt stress condition, but the level of up-regulation was more in PA4 compared with in PA2. As far as we know, no potential components of this cascade were

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known in P. australis plants. Our results indicated that the SOS signaling cascade may play an important role in protecting P. australis against salt stress. Therefore, an in-depth understanding of the SOS pathway and crucial kinases in the pathway may open up ways for the development of salt tolerant plants. Previous studies have shown that CDPKs may play critical roles in drought and salt stress signaling transduction and may act as important sensors of Ca2+ ion influx in the cytoplasm of plant cells in response to these stresses (Tuteja and Mahajan 2007). In this study, four unigenes encoding CDPKs (homologs of OsCPK12and OsCPK7) were up-regulated in PA4T plants, while their expression levels changed little in the PA2T plants. Over-expression of CDPK (OsCPK12) in rice has been reported to enhance tolerance to salt stress via scavenging the accumulation of ROS (Asano et al. 2011). Furthermore, in transgenic rice plants, OsCPK7 was induced by salinity and its up-regulation was reported to enhance salt tolerance (Saijo et al. 2000). Thus, modifying the expression of CDPKs in transgenic plants may be a way of enhancing tolerance to salt stress. Several studies have reported that the MAPK signaling cascade was involved in mediating drought, salinity, and cold stress tolerance in Arabidopsis (Teige et al. 2004), tomato (Wu et al. 2014), and Nicotiana benthamiana (Hashimoto et al. 2012). In the present study, numerous DEUs encoding MAP kinases, including MAPKKK2, MAPKKK1, MAPKK6, MAPK8, MAPK12, MAPK17, MAPK15, and MAPK2, were identified in the PA2 and PA4 plants. In PA4, these MAPK unigenes were significantly up-regulated, whereas, in PA2, some of them were down-regulated. These results suggested that the MAPK signaling pathway may also mediate salt stress signaling in P. australis. Recent studies have shown that MAPKK2 was detectable in Arabidopsis where it positively regulated salt and cold signaling transduction pathways (Qiu et al. 2008). Increased expression of the ZmMAPKK4 in maize was also found to enhance salt and cold tolerance (Wu et al. 2011). Xiong et al. (2002) suggested that an activated MAPK cascade can either directly activate transcription factors or can phosphorylate sensors and activators to continue downstream signal pathways. Thus, the detection of DEUs involved in MAPK signaling pathways in P. australis under salinity stress indicated that modulation of the signaling cascade may activate responsive transcription factors and protein kinases for enhanced salt tolerance in P. australis.

Conclusions The present study showed that diploid and autotetraploid P. australis exhibited significant differences in their responses to salinity stress. A series of morphological and physiological changes clearly demonstrated that normal metabolism was restrained in PA2 plants compared with PA4 plants under salinity stress. Transcriptome sequencing analysis revealed a

large number of unigenes that were differentially expressed in both PA2 and PA4. Some of the DEUs were confirmed by qRT-PCR. We found that many of the common unigenes involved in soluble carbohydrate and proline metabolism, ROS-scavenging system, ion transporters, and transcription factors showed contrasting expression patterns between the two accessions in response to salt stress. These findings reflected the physiological observations that PA2 suffered from greater negative effects than PA4 under salt stress conditions. The identified and, as yet, unidentified (novel) salt tolerance-related DEUs will provide important resources for candidate gene screening and further functional analyses for enhancing Paulownia salt tolerance. Funding information This work was supported by the Joint Funds of the National Natural Science Foundation of China (NSFC) (Grant No. U1204309), the Fund of the Transformation Project of the National Agricultural Scientific and Technological Achievement of China (Grant No. 2012GB2D000271), the Central Financial Forestry Science Promotion Project (Grant No. GTH [2012]01), the Fund of the Science Key Program of Department of Henan Education (Grant No. 12A220003), and the Fund of the Technology Innovation Team Project of Zhengzhou (Grant No. 121PCXTD515). Author contributions Guoqiang Fan conceived and designed the experiments, and supervised the study. Yanpeng Dong performed experiments, analyzed data, and wrote the manuscript. Yanpeng Dong revised the manuscript. Zhenli Zhao and Enkai Xu performed the experiments. Minjie Deng, Limin Wang and Suyan Niu contributed reagents or other essential material. Compliance with ethical standards Conflict of interest The authors declare that they have no competing interests. Data archiving statement Sequence data from this article have been deposited with the National Center for Biotechnology Information Sequence Read Archive database (http://www.ncbi. nlm.nih.gov/sra) under accession no. SRP059262.

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