Background

Salinity is one of the most severe abiotic factors threatening agricultural productivity and ecological environment throughout the world [2, 12]. Approximately half of irrigated lands in the world are threatened by salinity [43]. The expansion of soil salinization and the increasing human population are forcing agricultural production into marginal areas [44]. Soil salinization can significantly reduce the yield and quality of crops by resulting in a series of metabolic disturbances due to ionic toxicity, physiological drought and nutrient deficiency [14, 63]. Therefore, improving the salt tolerance of plants, especially traditional crops and forages, is currently an urgent issue, since most of these species are glycophytes with weak salt tolerance [43, 51]. In contrast, halophytic species have evolved multiple adaptation strategies to deal with harsh saline environments [12, 44]. Learning from halophytes, understanding the mechanisms underlying plant response to salinity and thereby identifying key genes related to salt tolerance will contribute to breeding crops with salt tolerance [61].

Halophytes can be divided into three types based on their adaptive strategies to saline environments: (i) pseudohalophytes maintain a low Na+ level by limiting Na+ uptake [52], (ii) euhalophytes compartmentalize Na+ into swollen internal vacuoles to alleviate Na+ toxicity in the cytosol [14, 56] and (iii) secretohalophytes exclude excessive Na+ from secreting structures (salt glands or salt bladders) on the surface of stems and/or leaves [11, 44]. For example, Reaumuria trigyna and Limonium bicolor are capable of secreting Na+ via their multicellular salt glands, but excreting little K+ to maintain high K+/Na+ ratio in the shoots [8, 62]. Mesembryanthemum crystallinum, Chenopodium quinoa and Atriplex species deposit a large amount of Na+ in epidermal bladder cells (EBCs) to improve salt tolerance of plants [26, 39]. Approximately half of all halophyte plants possess salt bladders, which segregate excessive Na+ away from metabolically active organs in the growing plant body [13, 44]; hence, these plants are likely to be potential species for saline soil amelioration and improvement of salt tolerance in important crops.

Atriplex canescens (four-wing saltbush), a C4 perennial semi-evergreen woody shrub with excellent adaptability to salinity and drought, is a typical secretohalophyte with salt bladders that is widely distributed in saline and arid regions [22]. This species is commonly planted in highway medians and on road shoulders, slopes, and other disturbed areas for erosion control and reclamation of marginal lands, and it can be used as a landscape plant in the arid regions of northern China; moreover, A. canescens is an attractive fodder crop for most livestock because of its high palatability and nutritional value [17, 40]. Early research findings showed that A. canescens could grow along a salinity gradient from 72 to 2017 mol/m3 NaCl in the root zone and accumulated more Na+ than K+ for osmotic adjustment (OA) at relatively low salinities [17, 18]. Our previous study revealed that moderate salinity (100 mM NaCl) could stimulate the growth of A. canescens and high salinity (400 mM NaCl) had no significant effect on its growth [40]. Under saline conditions, A. canescens can enhance photosynthetic capacity, accumulate more Na+ in tissues and salt bladders, maintain leaf K+ homeostasis, and use inorganic ions as well as organic osmolytes for OA, which may contribute to water balance in the plant [40]. Our latest investigation showed that the addition of 100 mM NaCl effectively alleviated the adverse impact of drought on the growth of A. canescens by increasing the accumulation of solutes (Na+, free proline, betaine and soluble sugar) in leaves as well as the net photosynthetic rate and water content (Guo H. and Bao A.K., unpublished data). All of these results indicate that the transport of Na+ and K+, the accumulation of organic solutes, the improvement of photosynthetic activity and leaf hydration are vital strategies for A. canescens adaptation to saline environments. Nonetheless, the possible molecular basis of these important physiological mechanisms is poorly understood owing to the absence of genomic data in A. canescens.

High-throughput RNA sequencing has been widely used to investigate the molecular processes related to adaptive responses to abiotic stresses and to identify stress-resistance candidate genes by analyzing differences in transcript abundance [57]. In this work, transcriptomes of A. canescens were generated by Illumina assembly technology to lay the foundation for exploring the potential salt tolerance mechanisms of this species. In addition, the genes showing significant transcriptional changes in A. canescens under NaCl treatment were then identified by comparing the gene transcript profiles in leaves and roots between salt-treated and control plants by using a tag-based digital gene expression (DGE) system, mainly focusing on identifying the candidate genes related to ion transport, organic osmolyte accumulation, water transport and photosynthesis.

Results

Transcriptome sequencing, de novo assembly and unigene functional annotation

A total of 13.37 and 13.41 Gb clean bases were generated from the leaves and roots of A. canescens by Illumina HiSeq sequencing, respectively (Additional file 1: Table S1). Then, 207.20 Mb raw reads were yielded from leaves and 210.00 Mb raw reads were yielded from roots through high-throughput sequencing (Additional file 1: Table S1). After filtering, a total of 133.70 and 134.12 Mb clean reads were generated from the leaves and roots, respectively, coupled with a Q20 score greater than 97 and 0.00% Ns (Additional file 1: Table S1). All of these results indicated that the output and quality of transcriptome sequencing were adequate for subsequent analysis.

Paired-end information was used to join contigs into scaffolds and further assembly23, and 54,611 and 59,582 unigenes, with a mean length of 912 and 696 bp, respectively, were generated from the leaves and roots (Table 1). Then, 70,571 all-unigene sequences were acquired, with a mean length of 961 bp, N50 of 1647 bp and GC percentage of 40.01%, after further assembly of the unigenes from leaves and roots (Table 1). The size distribution is shown in Additional file 1: Figure S1, and the lengths of 24,205 unigenes were more than 1000 bp.

Table 1 Overview of de novo sequence assembly
Full size table

Then, 44,121 unigenes (62.52% of the 70,571 unigenes) were annotated to known genes in 7 databases, namely, the Nr, Nt, Swiss-Prot, KEGG, COG, InterPro and GO databases (Additional file 1: Table S2). Functional annotation was not obtained for 37.48% of the unigenes due to the absence of genomic data in A. canescens and close-related species. Among these annotated unigenes, 26,021 unigenes annotated in the COG database in terms of sequence homology were classified into 25 functional clusters and 37,395 unigenes annotated with GO terms were grouped into 3 main GO categories with 52 subcategories (Additional file 1:Figures S2 and S3).

Differentially expressed genes (DEGs) in A. canescens under NaCl treatment

Eight independent cDNA libraries (CL6, CR6, SL6, SR6, CL24, CR24, SL24 and SR24) were sequenced, and approximately 22 million raw reads were generated in each library; after filtering low-quality reads, we obtained 21 million clean reads in each library (data not shown), more than 72% of which could be mapped to the transcriptome reference database (data not shown).

The DEGs in A. canescens were analyzed by comparing the 100 mM NaCl treatment with the control. When plants were subjected to the 100 mM NaCl treatment for 6 h, 14,686 and 16,306 DEGs were found in the leaves and roots, respectively, using the thresholds of FDR < 0.001 and |log2Ratio| > 1 (Fig. 1). Among these DEGs, 9023 and 4824 DEGs were upregulated, including 1768 and 1031 DEGs that were expressed in the leaves and roots of plants in the 100 mM NaCl treatment but almost not expressed in the control (the FPKM value was 0.01 in control plants, the same below), respectively (Fig. 1). Interestingly, the number of upregulated DEGs was much greater than that of downregulated DEGs in the leaves; conversely, the number of upregulated DEGs was much lower than that of downregulated DEGs in the roots. In addition, 3403 and 2405 DEGs (including 1799 and 1196 upregulated DEGs) were identified in the leaves and roots of plants treated to 100 mM NaCl for 24 h, respectively, which was far fewer than the number identified in plants exposed to the treatment for 6 h. Among these upregulated DEGs, 629 and 426 DEGs were expressed in the leaves and roots of plants in the 100 mM NaCl treatment while almost not expressed in the control, respectively (Fig. 1).

Fig. 1
figure 1

Numbers of differentially expressed genes (DEGs) in leaves and roots of A. canescens under 100 mM NaCl for 6 and 24 h. A FDR < 0.001 and an absolute value of the log2Ratio > 1 were used as the thresholds to determine significant differences in gene expression

Full size image

DEGs related to ion, glucose and oxygen transport

Firstly, the DEGs related to ion transport especially Na+ transport into salt bladders were identified under 100 mM NaCl treatment since accumulating a great quantity of Na+ in salt bladders and maintaining intracellular ion homeostasis are vital strategies for A. canescens adaptation to saline conditions.

In leaves, the number of upregulated DEGs associated with ion transport was much larger than that of downregulated DEGs when plants were treated with 100 mM NaCl for either 6 h or 24 h (Fig. 2). After plants were treated with 100 mM NaCl for 6 h, 76 DEGs were upregulated, which included important transcripts related to Na+ (including NHX and HKT), K+ (such as AKT and SKOR), Ca2+ (CNGC, CCX and P-Ca2+ ATPase), Mg2+ (MGT), and NH4+ (AMT) transport and the anion transport of NO3− (NRT), PO43− (PHT), SO42− (STAS), Cl− (CLC and SLAH) and several important micronutrients (such as Zn, Mo, B and Cu) (Fig. 2a; Additional file 1: Table S3). Some genes encoding plasma membrane H+-ATPases (P-H+ ATPase) and vacuolar H+-pyrophosphatases (V-H+ PPase) were also upregulated (Fig. 2a). The number of DEGs was significantly lower under 100 mM NaCl for 24 h than under 100 mM NaCl for 6 h (Fig. 2b). Among these upregulated genes, two transcripts associated with Na+ transport (SOS1 and HKT1) were upregulated, and the other upregulated DEGs were mainly SKOR, AKT, CNGC, MGT and NRT transport protein family genes, which are related to nutrient element transport (Fig. 2b; Additional file 1: Table S4). In addition, among these DEGs related to K+ transport in leaves, 9 DEGs were significantly upregulated under 100 mM NaCl but not under control conditions for either 6 or 24 h (Table 2).

Fig. 2
figure 2

The DEGs related to ion transport in leaves of A. canescens under 100 mM NaCl for 6 (a) and 24 h (b). NHX: tonoplast Na+/H+ antiporter, SOS1: plasma membrane Na+/H+ antiporter, HKT: high-affinity K+ transporter, KEA: K+ efflux antiporter, KCO: calcium-activated outwardly rectifying potassium channel, SKOR: stelar K+ outwardly rectifying channel, AKT: inwardly rectifying K+ channel, KT/HAK/KUP: K+ transporter, CNGC: cyclic nucleotide-gated channel, CCX: cation/Ca2+ exchanger, P-Ca2+ ATPase: plasma membrane Ca2+ ATPase, V-CAX: vacuolar cation/H+ exchanger, P-H+ ATPase: plasma membrane H+ ATPase, V-H+ PPase: vacuolar H+ PPase, MGT: Mg2+ transporter, AMT: NH4+ transporter, NRT: NO3− transporter, STAS: SO42− transporter, PHT: PO43− transporter, CLC: vacuolar Cl−/H+ exchanger, SLAH: Slow type anion channel, CTR: Cu2+ transporter, BOR: BO3− transporter, ZnT: Zn3+ transporter, MOT: MoO42− transporter. The up and down arrows indicate the total number of up and downregulated DEGs, respectively. The same below

Full size image
Table 2 The upregulated DEGs related to K+ transport in roots and leaves of A. canescens under 100 mM NaCl but not under control condition for either 6 or 24 h
Full size table

The epidermal bladder cell (EBC) together with stalk cell (SC) and epidermal cell (EC) constitute the EC-SC-EBC complex [44]. The Na+ sequestration in the high vacuolization of salt bladder is achieved by four times Na+ transport through plasma membrane and one time Na+ transport through tonoplast [37]; furthermore, tomato regulates the activity of these enzymes by controlling the expression patterns of their genes to adapt to salinity [30]. Amylase (AMS), mannitol dehydrogenase (MD) and trehalose-6-phosphate synthase (TPS) play indispensable roles in starch, mannitol and trehalose metabolism, respectively, and their enzyme activities are positively related to the salt tolerance of plants [7]. In this study, multiple genes encoding key enzymes for the synthesis of proline, betaine and soluble sugar in leaves were upregulated under 100 mM NaCl (Fig. 5; Additional file 1: Tables S7-S8), and 13 genes (such as P5CS, BADH and SPS) of these upregulated genes were continuously expressed under salt treatment for both 6 and 24 h (Table 3), suggesting that A. canescens possesses an efficient mechanism for accumulating osmoprotectants under saline conditions by modulating the expression patterns of important genes involved in compatible solute biosynthesis, which would be conducive to protecting plants in saline environments by OA.

Aquaporin plays an important role in the regulation of water balance in A. canescens under saline conditions

A. canescens seedlings can maintain a higher leaf relative water content by effective OA under salinity treatment [40]. Moreover, our latest study found that water was abundantly transported as a solvent into salt bladders with the accumulation of Na+ in salt bladders of A. canescens under NaCl treatment, causing rapid expansion of the salt bladders, a sharp increase in turgor pressure and eventually the bursting of the bladders and subsequent release of a large amount of accumulated Na+ (Guo H. and Bao A.K., unpublished data); this finding suggests that the rapid accumulation of water in salt bladders is the key factor affecting salt secretion in A. canescens. AQPs can effectively regulate the water balance inside and outside of the plant cell by specifically mediating the rapid transmembrane transport of water [6]. In this study, many DEGs related to AQPs were upregulated in leaves but not in roots under 100 mM NaCl for 6 h, including 6 nodulin-like intrinsic proteins (NIPs), 1 small basic intrinsic protein (SIP), 2 plasma membrane intrinsic proteins (PIPs) and 3 tonoplast intrinsic proteins (TIPs) (Table 4). The NIPs mainly mediate boron uptake (NIP5;1) or are involved in pollen development and pollination (NIP4) in Arabidopsis thaliana [10, 55]. The SIPs are localized to the endoplasmic reticulum membrane but currently are not well characterized [25]. Plasma membrane-located PIPs are divided into the PIP1 and PIP2 groups; PIP2 members mainly function as water channels, while PIP1 members usually have much lower or no water conductivity due to their failure to localize to the plasma membrane [5]. Our results showed that the transcript of 1 PIP gene (CL969.Contig2_All, highly homologous to PIP2;3) was significantly upregulated in leaves (Table 4). PIP2 in Malus zumi Mats is involved in water movement during both water absorption and transport and alters the salt tolerance of transgenic Arabidopsis [53]. An AQP protein, AcPIP2, characterized from A. canescens, improved plant growth rate and salt tolerance when overexpressed in A. thaliana [28]. Moreover, tonoplast-located TIPs, primarily mediating the accumulation of water in the vacuole, play vital roles in maintaining cell turgor and enhancing the capacity for OA and are also able to indirectly promote Na+ compartmentation into vacuoles, which is conducive to improving plant adaptation to saline environments [25, 36]. In the present study, we found that the transcripts of 3 TIPs (Unigene15728_All, highly homologous to TIP2;2; Unigene12348_All, highly homologous to TIP4;1 and CL5261.Contig2_All, highly homologous to TIP1;3) were sharply upregulated in the leaves but downregulated in the roots of A. canescens under NaCl treatment, and there was even a 14.24-fold increase in the transcript level of AcTIP2;2 (Table 4). Therefore, the AcPIP2 and AcTIPs in the leaves of A. canescens are likely to be involved in the transport of water into salt bladders under salt treatment, which facilitates salt secretion and the maintenance of the water balance in leaves and might result from the accumulation of solutes in leaf tissues and salt bladders.

Moderate salinity improves photosynthesis of A. canescens by increasing the transcripts of photosynthesis-related genes

Previous studies showed that NaCl significantly improved the photosynthetic capacity of A. canescens plants, and the trends of photosynthetic indicators were different from those of C3 xerohalophytes, suggesting that Na+ may promote the C4 photosynthetic process of A. canescens under saline conditions [40]. C4 plants share stronger CO2 assimilation capacity and can sufficiently utilize light energy; moreover, this kind of plant have greater adaptability to adversity since they can take advantage of low CO2 levels in the intercellular space under stress conditions [15, 27]. The oxygenic photosynthesis of higher plants can be divided into three stages: the primary reaction, photosynthetic electron transport and photophosphorylation and CO2 assimilation. The first two steps in this process involve the conversion of sunlight into active chemical energy, which is driven by several multisubunit membrane protein complexes, including photosystem II, cytochrome b6/f, photosystem I, ferredoxin and ATP synthase [38]. The last reaction is a process that converts CO2 into stable chemical energy stored in organic matter by using the energy (ATP and NADPH) produced by the light reaction [15]. Our study found that the majority of DEGs related to the above-mentioned complexes and the enzymes involved in carbon fixation and chlorophyll biosynthesis/catabolism were downregulated under 100 mM NaCl for 6 h (Fig. 7a, Additional file 1: S13), but the few upregulated genes were mainly involved in electron transport and carbon fixation, the latter of which were almost always involved in the C4 pathway, including phosphoenolpyruvate carboxylase (PEPC), malate dehydrogenase (MDH), malic enzyme (ME) and aspartate aminotransferase (AST) (Table 5). These enzymes play key roles in the C4 photosynthetic pathway and are capable of providing more CO2 for the C3 pathway in the vascular bundle sheath61. Interestingly, the transcript levels of many DEGs involved in the processes of chlorophyll biosynthesis, electron transport and carbon fixation (based on the C3 pathway) were significantly upregulated after treatment with 100 mM NaCl for 24 h but not after treatment for 6 h (Table 5), suggesting that A. canescens preferentially increased the transcript abundances of genes encoding key enzymes in the C4 pathway to improve its assimilation capacity and then increased the transcript levels of other genes encoding complexes related to chlorophyll biosynthesis, electron transport and C3 carbon fixation under salt treatment, which might be one of the important reasons the photosynthesis of A. canescens was significantly improved by 100 mM NaCl [40]. At present, re-engineering C3 plants with C4 CO2-concentrating mechanisms is of broad interest [34, 45]. Overexpression of plastidic ZmNADP-MDH (NADP-MDH) in maize conferred salt tolerance to transgenic Arabidopsis [24]. Thus, the results of this study provide abundant genetic resources for improving photosynthetic efficiency in C3 crops/forages through genetic engineering.

Conclusions

This study identified candidate genes showing significant transcriptional changes in A. canescens under 100 mM NaCl treatment, mainly focusing on genes related to ion transport, organic osmolyte synthesis, water transport and photosynthesis. The abundance of transcripts encoding transporters/channels for important macro- and microelements was significantly increased by 100 mM NaCl, which is conducive to promote the uptake and transport of nutrient elements. It is worth noting that some genes related to Na+ transport in leaves (such as AcSOS1, AcHKT1 and AcNHX) might play crucial roles in the excretion of salt via epidermis bladder cells. In addition, the transcripts of a number of genes related to the synthesis of organic osmolytes in leaves was significantly upregulated by NaCl treatment, which allowed the accumulation of more organic solutes to enhance OA under salt treatment. Moreover, 100 mM NaCl promoted water transport in A. canescens by inducing the transcripts of aquaporin-encoding genes in leaves. Interestingly, NaCl preferentially induced the transcripts of genes encoding proteins participating in the C4 photosynthetic pathway to provide greater assimilation capacity for photosynthesis and then increased the transcript levels of other genes encoding complexes related to chlorophyll biosynthesis, electron transport and C3 carbon fixation under salt treatment. Our results lay the foundation for investigating molecular mechanisms of salt tolerance in secretohalophytes and provide a theoretical basis for genetic improvement of stress tolerance in important crops and forages by using the outstanding gene resources from A. canescens.

Methods

Plant materials and experimental treatments

Seeds of Atriplex canescens were collected from Lingwu County in Ningxia Autonomous Region, China. After removed the hard seed coat with 75% H2SO4 (v/v) for 15 h, the seeds were washed many times with purified water until the washings has no smell and then germinated in moist vermiculite at 28 °C in the dark for 5 days. Uniform seedlings were transplanted into plastic containers (5 cm × 5 cm × 5 cm; two plants/pot) filled with vermiculite and irrigated with 1/2-strength Hoagland nutrient solution at 2-day intervals [40]. Plants were cultured at 28 °C/25 °C (day/night), 16/8 h photoperiod (light/dark; the light density was approximately 800 μmol/m2/s) and 65% relative humidity.

Four-week-old seedlings were treated with 1/2-strength Hoagland nutrient solution supplemented with 0 (control) or 100 mM NaCl. The leaves and roots of seedlings in the two treatments were collected after treatment for 6 and 24 h, respectively. A total of eight samples were marked as follows: CL6, CR6, SL6, SR6, CL24, CR24, SL24 and SR24; C and S represent the control and treatment with 100 mM NaCl, respectively; 6 and 24 denote the treatment duration; and R and L denote the roots and leaves, respectively. For example, SL6 and SR6 were the leaf and root samples, respectively, from salt-treated plants for which seedlings were treated for 6 h. All the fresh samples were immediately frozen in liquid nitrogen and stored at − 80 °C until RNA extraction.

RNA preparation, cDNA library construction and Illumina sequencing

Total RNA was isolated from the eight samples with an RNeasy Plant Mini Kit (Qiagen). The extracted RNA was quantified by using a NanoDrop ND-1000 instrument (Thermo Scientific), and the integrity of the RNA was determined by 1% agarose gel electrophoresis. Equivalent amounts of total RNA isolated from each of the four leaf tissues (CL6, SL6, CL24 and SL24) and each of the four corresponding root tissues (CR6, SR6, CR24 and SR24) were pooled. The two mRNA pools were then used for reverse transcription to obtain two cDNA libraries as the cDNA in the leaves and roots of A. canescens by using the method described by Dang et al. [8] and sequenced on an Illumina HiSeq™ 2000 platform in BGI Shenzhen.

De novo assembly and functional annotation

High-quality clean reads were created after filtering adaptor sequences, duplicated sequences, reads containing more than 5% ambiguous bases (‘N’) and reads in which more than 50% of bases showed a Q-value ≤5. After filtering out low quality reads, de novo assembly was proceeded by using Trinity; then, the Trinity unigenes were clustered with TGICL software to minimize sequence redundancy [21]. The unigenes were divided into two classes after performing gene family clustering, one class included clusters with the prefix CL contained several unigenes with a sequence similarity of more than 70%, and the other included singletons with the prefix unigenes [31]. To attach predicted gene informations for assembled unigenes, the sequences were functionally annotated based on seven protein databases (including the Nr, Nt, Swiss-Prot, Kyoto Encyclopedia of Genes and Genomes (KEGG), Clusters of Orthologous Groups (COG), InterPro and Gene Ontology (GO) databases) using the BLASTX tool with an E-value ≤10− 5 threshold. Blast2GO software was employed to perform functional categorization by GO terms on the basis of biological process, cellular component and molecular function ontologies, and the Web Gene Ontology Annotation Plot (WEGO) tool was used to statistically analyze the data [8].

Differentially expressed gene (DEG) library preparation and analysis

Eight independent cDNA libraries (CL6, CR6, SL6, SR6, CL24, CR24, SL24 and SR24) were prepared in parallel for leaves and roots at different times under salt treatment by using a tag-based DGE kit [59]. Then, each library was sequenced through the Illumina HiSeq™ 2000 sequencing platform in BGI Shenzhen. After low-quality reads (including reads with adaptors, more than 10% unknown nucleotides (‘N’) and only one copy number) were removed, the clean reads were mapped to the transcriptome reference database. And then, the transcript levels of all assembled unigenes were calculated by using the number of fragments per kb per million reads (FPKM) method to identify differentially expressed genes (DEGs) [59]. In addition, the false discovery rate (FDR) method was used to confirm the threshold P-value for multiple tests and analysis by manipulating the FDR value. An FDR < 0.001 and an absolute value of |log2Ratio| > 1 were used as thresholds to identify DEGs [8].

qRT-PCR validation of DEGs

To experimentally evaluate the RNA-Seq results, total RNA was extracted from the 8 samples as described before and reversely transcribed into cDNA according to the manufacturer’s protocol (TaKaRa Biotechnology). The qRT-PCR was conducted by using SYBR Green Real-Time PCR Master Mix (TaKaRa Biotechnology) and performed on a StepOnePlus Real-Time PCR Thermocycler (Applied Biosystems, USA). ACTIN gene was used as the internal standard. The relative transcript levels of the 30 randomly selected unigenes were calculated using the 2-ΔΔCt method [31].