Transcriptomic profiling identifies candidate genes involved in salt tolerance of the xerophyte Pugionium cornutum

doi:10.21203/rs.2.14265/v1

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Transcriptomic profiling identifies candidate genes involved in salt tolerance of the xerophyte Pugionium cornutum

https://doi.org/10.21203/rs.2.14265/v1

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published 12 Dec, 2019

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Background: Pugionium cornutum is a xerophytic plant that primarily adapts to salt stress by accumulating inorganic ions (e.g., Cl-) for osmoregulation, improving its reactive oxygen species (ROS)-scavenging ability and maintaining high photosynthetic carbon assimilation efficiency, but the associated molecular mechanisms still remain unclear.

Results: Here, we present an analysis of gene responses to salt stress based on the transcriptome of P. cornutum exposed to 50 mM NaCl treatment. The data revealed that, after NaCl treatment for 6 or 24 h, the transcript levels of multiple genes encoding proteins facilitating Cl- accumulation and NO3- homeostasis such as SLAH1, CLCg, CCC1, and NPF6.4, as well as the transport of other major inorganic osmoticums were significantly upregulated in roots and shoots, which should be favorable to enhancing osmotic adjustment capacity and maintaining the plant uptake and transport of nutrient elements; a large number of genes related to ROS-scavenging pathways were also significantly upregulated, which should be beneficial for mitigating salt-induced oxidative damage to the cell metabolism. Meanwhile, many genes encoding components of the photosynthetic electron transport and carbon fixation enzymes were significantly upregulated in shoots after salt treatment, possibly resulting in a high carbon assimilation efficiency in P. cornutum. Additionally, numerous salt-inducible transcription factor genes probably regulating the abovementioned processes were found.

Conclusion: Candidate genes involved in salt tolerance of P. cornutum were identified, which lays a preliminary foundation for clarifying the molecular mechanism of the xerophytes adapting to harsh environments.

Plant Physiology and Morphology

Plant Molecular Biology and Genetics

Salt tolerance

transcriptome

differentially expressed genes

xerophyte

Pugionium cornutum

Soil salinity is one of the primary abiotic stresses that limits the sustainable development of agriculture worldwide, and approximately one-fifth of the world’s cultivated land has been affected by salinity [1–3]. Salt tolerance in plants is a complex trait involving responses to cellular osmotic and ionic stresses and their consequent secondary stresses (e.g., oxidative stress), which are all polygenic processes [4]. Most conventional and staple crop and forage species are very sensitive to high concentrations of salt in soils and have limited genetic potential for salt tolerance because of their long-term growth under favorable cultivation conditions [5, 6]. The breeding of crops with higher yield and quality under saline conditions by using molecular genetics is an effective strategy to promote food security [3, 7, 8]. Despite abundant molecular knowledge have derived from model plantssuch as Arabidopsis thaliana and Oryza sativa, a low capacity for stress tolerance limits their usefulness in current breeding programs [9]. Certain wild plant species, including halophytes and xerophytes, however, have evolved multiple protective mechanisms to successfully thrive in extremely harsh environments and, as a result, harbor prominent abiotic stress tolerance genes [10, 11]. Hence, it is possible to genetically improve salt tolerance in crops and forage through a better understanding of the molecular basis underlying the adaptive strategies to soil salinity employed by these halophytes and xerophytes [6, 12].

Pugionium cornutum, a xerophytic desert plant in the genus Pugionium Gaertn belonging to Brassicaceae, is primarily found in arid and semi-arid regions of northwestern China, with strong adaptability to various environmental stresses including salinity and drought [13, 14]. In local areas, this species plays an important role in sand fixation and soil and water conservation due to its well-developed root systems; moreover, the higher medicinal property and nutrient content make it attractive as a traditional Chinese herb and vegetable [14, 15]. Previous studies have found that P. cornutum possesses a strong ability to reduce oxidative damage under salt and drought stresses by increasing the activities of enzymes involved in reactive oxygen species (ROS) scavenging such as superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) [16–18]. It was also demonstrated that P. cornutum is a typical Cl^--tolerant species and can tolerate high tissue Cl^- concentrations that can be even toxic to some other Cl^--tolerant species; moreover, P. cornutum can directly use Cl^- as an efficient and beneficial osmoticum for enhancing its leaf turgor and hydration under salt stress [19, 20]. Meanwhile, a substantially increased accumulation of other major inorganic osmoticums, such as Na⁺, SO₄^2-, and PO₄^3-, as well as relatively stable concentrations of K⁺ and NO₃^-, were observed in roots and shoots of P. cornutum under NaCl treatments [19, 20]. Therefore, the large absorption of inorganic osmoticums, especially Cl^-, and the maintenance of K⁺ and NO₃^- homeostasis are vital physiological mechanisms in the adaptation of P. cornutum to salt stress. In addition, the dry biomass of P. cornutum did not decrease under 50 mM NaCl stress despite the significantly suppressed leaf net photosynthetic rate [19], indicative of a high carbon assimilation efficiency of P. cornutum under salt stress. Although these physiological traits in salt tolerance of P. cornutum have been documented, the corresponding molecular foundation remains elusive and should be investigated to identify the prominent genes involved. Furthermore, to date, studies on the molecular mechanisms involved in the ability of higher plants to tolerate Cl^--toxicity continue to lag behind and are mainly focused on the model plant A. thaliana and Cl^--sensitive crops such as soybean, citrus and grape [21]. Thus, understanding on the molecular basis in P. cornutum coping with Cl^--toxicity would help to further elucidate Cl^- tolerance in higher plants.

RNA sequencing (RNA-Seq) is a rapid and cost-effective method for both mapping and quantifying the transcriptome in eukaryotes and is widely used to analyze the gene expression in plants under specific developmental stages or physiological conditions, providing substantial insights into the molecular processes involved in plants’ response to abiotic stresses [9, 22]. In the present work, we generated a transcriptomic dataset for studying the mechanisms of salt tolerance in P. cornutum using Illumina high-throughput sequencing technology. Then, we analyzed the differentially expressed functional genes related to ROS scavenging, ion (especially Cl^-) transport, and photosynthesis as well as transcription factor genes under 50 mM NaCl treatment by using a tag-based digital gene expression (DGE) profiling technique.

Transcriptome sequencing, de novo assembly and functional annotation

Transcriptome sequencing generated more than 119 and 118 million raw reads from the roots and shoots of P. cornutum, respectively (Supplementary Table S1). After exclusion of the low-quality reads, including empty reads, adapter reads and reads with unknown nucleotide “N” or only one copy number, 117 and 116 million clean reads with GC percentage of 45% and 46% were obtained from the roots and shoots, respectively (Supplementary Table S1). The total clean reads/total raw reads percentages were more than 98% for both roots and shoots. Only a small number of reads was excluded by filtering, indicating that the data were highly reliable.

The de novo assembly of total clean reads generated 64,978 unigenes from shoots and 80,307 unigenes from roots, with an average mean length of 1091 and 997 bp, respectively (Table 1). After further assembly and redundancy elimination of these unigenes using the CAP Assembler, 72,068 gap-free unigenes with an average length of 1243 bp were obtained (Table 1). The length distribution of all unigenes showed that the length of 34,108 unigenes (more than 50%) was greater than 1000 bp (Supplementary Fig. S1).

In total, 63,396 unigenes were matched to known homologs from other plant species by being aligned against the protein databases including NR, NT, Swiss-Prot, KEGG, COG, GO, accounting for 88% of the total unigenes (Supplementary Table S2). Specifically, 57,695, 60,628 39,893, 35,136, 24,608 and 53,435 unigenes had functional annotations in the above six databases, which accounted for 80, 84, 55, 49, 34 and 74% of the total unigenes, respectively (Supplementary Table S2).

Identification of DEGs in the roots and shootsof P. cornutum treated with 50 mM NaCl

After treatment with 50 mM NaCl for 6 h, 7356 and 7088 differentially expressed genes (DEGs) were identified in P. cornutum roots and shoots, respectively, among which 4020 and 3673 were upregulated, and 3336 and 3415 were downregulated, respectively (Fig. 1). After treatment with 50 mM NaCl for 24 h, 12,648 and 4905 DEGs were identified in the P. cornutum roots and shoots, among which 4064 and 1972 were upregulated and 8584 and 2933 were downregulated, respectively (Fig. 1).

DEGs related to ion transport

The absorption and accumulation of inorganic osmoticums such as Cl^-, Na⁺, SO₄^2- and PO₄^3-, as well as maintaining homeostasis of the inorganic macronutrients K⁺ and NO₃^-, are primary physiological mechanisms in the salt tolerance of P. cornutum [19, 20]. Therefore, we firstly analyzed the expression changes in DEGs related to ion transport in the roots and shoots of P. cornutum treated with 50 mM NaCl for 6 and 24 h.

After treatment with 50 mM NaCl for 6 and 24 h, 49 and 41 DEGs associated with Cl^-, NO₃^-, Na⁺, K⁺, Ca²⁺, SO₄^2-, PO₄^3-, Zn²⁺, Cu²⁺, NH₄⁺, Mn²⁺, Mg²⁺, Fe²⁺, BO₃^3- and H⁺ transport were respectively upregulated in roots, and 18 and 31 DEGs were respectively downregulated in roots (Fig. 2A and B). Among the upregulated DEGs, a large proportion was Cl^- and/or NO₃^- transporter-encoding genes, including SLAH1, CLCs and NPFs. It was noteworthy that the transcript levels of SLAH1, CLCg, NPF6.4 were upregulated in roots after treatment with 50 mM NaCl for both 6 and 24 h (Table 2, Supplementary Table S3 and S4), indicating that these genes might be closely involved in Cl^- absorption and accumulation, as well as NO₃^- homeostasis of P. cornutum under saline conditions. Furthermore, it was observed that SLAH1 was almost completely unexpressed in roots under the control condition (the RPKM value of SLAH1 was 0.01 under the control condition for both 6 and 24 h), while its expression level was increased by over 8-fold after salt treatment for both 6 and 24 h (Supplementary Table S3 and S4), indicative of a vital role of SLAH1 in salt response of P. cornutum. Among the downregulated DEGs related to Cl^- and/or NO₃^- transport, the transcript abundance of CCC1, which encodes a Cl^- transporter facilitating the retrieval from xylem sap into roots [23], was substantially decreased in roots under salt treatment for 24 h (Supplementary Table S4). Na⁺/H⁺ antiporters (NHXs) are widely recognized to play important roles in Na⁺ and/or K⁺ homeostasis in plants under saline conditions [24–26]. Our results showed that the transcript abundance of plasma membrane-located Na⁺/H⁺ antiporters-encoding gene SOS1 (also named NHX7) in roots was upregulated after salt treatment for 6 h but downregulated after salt treatment for 24 h (Fig. 2A and B, Supplementary Table S3 and S4), indicating that SOS1 in roots might positively regulate salt tolerance in P. cornutum during relatively short-term salt treatment. The transcript abundance of the vacuole-located Na⁺/H⁺ antiporters-encoding gene NHX1 was upregulated in roots after 24 h of salt treatment, and the golgi-located Na⁺/H⁺antiporters-encoding genes NHX5 and NHX6 were upregulated after both 6 and 24 h of salt treatment (Fig. 2A and B, Supplementary Table S3 and S4); therefore, these three genes very likely confer Na⁺/K⁺ homeostasis in roots of P. cornutum under saline conditions. Furthermore, several K⁺ transporter-encoding genes that also contribute to Na⁺/K⁺ homeostasis, such as KT2, KT3, KUP2, HAK3, KEA1, KEA4, KEA5 and KEA6, were upregulated under salt treatment for either 6 or 24 h (Fig. 2A and B, Supplementary Table S3 and S4). CNGCs are important plasma membrane-located cation channels mainly permeable to K⁺, Na⁺ and Ca²⁺; GLRs as glutamate (Glu) receptors can provide Glu to activate plasma membrane-located nonspecific cation channels in root cells and, therefore, trigger large and rapid rises in the concentrations of cytosolic cations, especially Ca²⁺ [27–29]. Our results also characterized many upregulated CNGC and GLR genes in roots of P. cornutum under salt treatment (Fig. 2A and B), indicating that P. cornutum is also likely to maintain root cationic homeostasis under saline conditions by increasing the expression of these CNGC and GLR genes in roots to control K⁺, Na⁺ and Ca²⁺transport across the cell membrane. Many upregulated DEGs related to the transport of other inorganic ions, including SO₄^2- (e.g., Sultr1.2, Sultr2.1; Sultr3.1),, PO₄^3- (e.g., PhT1.3, PhT2.1, PhT4.3, PHO1),, Zn²⁺ (e.g., ZnT1, ZnT8, ZnT12),, Cu²⁺ (e.g., CTR2, CTR5, CCH, PAA1),, Mn²⁺ (e.g., PDR2),, Fe²⁺ (e.g., IRT1),, Mg²⁺ (e.g., MgT),, NH₄⁺ (e.g., AMT1.1, AMT1.2, AMT2),, and BO₃^3-(e.g., BOR3, BOR4),, were also identified in roots of P. cornutum under either 6 or 24 h salt treatment (Fig. 2A and B, Supplementary Table S3 and S4). Additionally, as the transport of inorganic ions across cell membranes generally couples H⁺ as a proton motive force [30], the upregulated P-H⁺ ATPase and V-H⁺ ATPase in roots under salt treatment identified by our data (Fig. 2A and B) likely play essential roles in ion transport across the root cell plasma membrane and tonoplast, in addition to maintaining the cellular membrane electrochemical gradient.

After salt treatment for 6 and 24 h, 46 and 30 DEGs in shoots associated with inorganic ion transport were respectively upregulated and 23 and 26 DEGs in shoots were respectively downregulated (Fig. 2C and D). In contrast to the observation in roots, the expression of CCC1 in shoots was considerably upregulated under both 6 and 24 h of salt treatment (Fig. 2C and D, Supplementary Table S5 and S6), which might be beneficial for the exclusion of more Cl^- from shoot vascular tissues to facilitate Cl^- uptake by mesophyll cells. Cl^- can only be largely accumulated in plant shoots by compartmentalization into vacuoles of mesophyll cells [31, 32]. This process in higher plants is dominated by the tonoplast-located chloride channel CLCg [33]. Our results showed that the transcript abundance of CLCg in shoots was upregulated under salt treatment for both 6 and 24 h (Table 3). Hence, CLCg should play a key role in the Cl^--accumulating characteristic of P. cornutum under saline conditions. In addition to CLCg, CLCb, which encodes tonoplast NO₃^- transporter [34], and several NPFs such as NPF6.3 and NPF6.4 that encode NO₃^- and/or Cl^- transporters [35], were also upregulated under salt treatment (Supplementary Table S5 and S6), suggesting that these genes might also be essential for Cl^- accumulation and NO₃^- homeostasis in shoots of P. cornutum under saline conditions. Certain DEGs found in roots encoding Na⁺/H⁺ antiporters such as SOS1 and NHX1; K⁺ transporters such as KT2, KUP2, KEA1, KEA4, KEA6; cation channels such as CNGC1 and CNGC6; and glutamate receptors such as GLR3.3, were also upregulated in shoots after salt treatment for either 6 or 24 h (Supplementary Table S5 and S6), indicating that these genes might also regulate Na⁺/K⁺ homeostasis in shoots of P. cornutum under saline conditions. Furthermore, several DEGs related to Na⁺ or K⁺ transport, including HKT1, SKOR and GORK, were only found in shoots but not in roots after salt treatment (Fig. 2). HKT1-type proteins facilitate Na⁺ absorption and transport in the roots of model plants A. thaliana [36, 37]. But, in P. cornutum, HKT1 is likely to be distinctively responsive to salt stress in shoots. As an outward rectifying K⁺ channel, GORK has been proven to participate in stomatal movement [38]; therefore, the observed upregulation of GORK in shoots after salt treatment (Fig. 2D) are likely essential for stomatal movement and, consequently, affect salt tolerance in P. cornutum. Many DEGs related to the transport of other inorganic nutrients or osmoticums, such as SO₄^2-, Zn²⁺, Cu²⁺, Mn²⁺, Fe²⁺, Mg²⁺, and NH₄⁺, were identified in shoots after salt treatment for either 6 or 24 h (Fig. 2C and D), indicating that these genes might also regulate shoot osmotic adjustment of P. cornutum under saline conditions. Among the DEGs encoding V-H⁺ ATPase, P-H⁺ ATPase and P-Ca²⁺ ATPase in shoots, V-H⁺ ATPase c2, V-H⁺ ATPase e1 and V-H⁺ ATPase h were not expressed under the control condition but were highly expressed under salt treatment (Supplementary Table S5 and S6), suggesting that these three genes might play vital roles in vacuole compartmentation of inorganic osmoticums for facilitating shoot osmotic adjustment.

DEGs related to ROS-scavenging system

The ROS-scavenging system in higher plants mainly consists of the ascorbate-glutathione (AsA-GSH) cycle, glutathione peroxidase (GPX) pathway, catalase (CAT) pathway and peroxiredoxin/thioredoxin (PrxR/Trx) pathway [9]. After salt treatment for 6 and 24 h, a total of 19 and 15 DEGs categorized in the abovementioned ROS-scavenging pathways in roots of P. cornutum were respectively upregulated and 5 and 29 DEGs were respectively downregulated (Fig. 3A and B). Among the upregulated DEGs in roots after 6 h of salt treatment, most were GSTs (involved in both the ASA-GSH and GPX pathways) and Trxs (involved in the PrxR/Trx pathway) (Fig. 3A). Hence, the ASA-GSH, GPX and PrxR/Trx pathways might be the major components of the root ROS-scavenging system of P. cornutum in adaption to salinity. After 24 h of salt treatment, although many DEGs associated with ROS scavenging were downregulated, the expression of GST-U10 (no expression under the control condition) and TrxH8 was still obviously upregulated (Fig. 3B, Supplementary Table S8), indicating that these two genes might play vital roles in ROS scavenging in roots of P. cornutum under saline conditions. Furthermore, after salt treatment for 24 h, 2 upregulated PODs, 1 upregulated CAT and 1 upregulated SOD emerged (Fig. 3B), indicating that these genes might mainly facilitate root ROS-scavenging of P. cornutum during relatively long-term salt treatment.

Following 6 h of 50 mM NaCl treatment, the expression levels of 3 GST genes and 2 GPX genes in the GPX pathway were upregulated in the P. cornutum shoots (Fig. 3C). In particular, GST-F9 showed no expression under the control condition, with high expression detected in the shoots after 6 h of 50 mM NaCl treatment (Supplementary Table S9). Despite the finding that fewer POD genes were upregulated than downregulated, one POD (POD43) was not expressed under the control condition, whereas its expression was substantially induced in the shoots after 6 h of 50 mM NaCl treatment (Fig. 3C, Supplementary Table S9). Almost all the differentially expressed PEX, Trx and PrxR genes in shoots after 6 h of salt treatment were upregulated (including 1 PEX and 2 Trxs, which were not expressed under the control condition but were highly expressed under salt treatment) (Fig. 3C, Supplementary Table S9), indicating that the CAT and PrxR/Trx pathways should mainly function in the shoot ROS-scavenging system of P. cornutum under salt treatment. Following 24 h of salt treatment, only a few genes associated with ROS scavenging were differentially expressed in shoots, and most of them were downregulated (Fig. 3D). However, PEX3–1a showed no expression under the control condition but upregulated expression under both 6 and 24 h of salt treatment (Supplementary Table S9 and S10), suggesting that PEX3–1a might play a vital role in ROS scavenging in shoots of P. cornutum under saline conditions.

DEGs related to photosynthesis

The maintenance of high carbon assimilation efficiency is an important strategy by which P. cornutum adapts to soil salinity [19]. The oxygenic photosynthesis of higher plants consists of photosynthetic electron transport with many components such as chlorophyll, photosystem II-light harvesting complex (PS II), photosystem I-light harvesting complex (PS I), cytochrome b₆f complex, ferredoxin, ATP synthase, and carbon fixation using various enzymes [39]. Thus, we analyzed the expression of DEGs related to the abovementioned processes in P. cornutum treated with 50 mM NaCl.

Under 50 mM NaCl treatment for 6 h, the majority of DEGs in shoots of P. cornutum related to the components of PS II complex and chlorophyll biosynthesis were upregulated and all of the DEGs related to the components of PS I complex, cytochrome b₆f complex and ATP synthase were upregulated (Fig. 4A), suggesting that the expression of these genes in shoots might be essential for light energy absorption and photosynthetic electron transport to generate more ATP for carbon fixation. Furthermore, it was observed that nearly 20 DEGs encoding various enzymes (mainly malate dehydrogenase and phosphoglycerate kinase) in carbon fixation process were upregulated (Fig. 4A, Supplementary Table S11), indicative of a vital role of these genes in maintaining high carbon assimilation efficiency in P. cornutum under salt stress.

Under 50 mM NaCl treatment for 24 h, the numbers of upregulated DEGs in shoots related to the components of PS II complex, PS I complex, chlorophyll biosynthesis and ATP synthase were much lower than that under 50 mM NaCl treatment for 6 h, but the expression of one cytochrome f-encoding gene, one ferredoxin-dependent glutamate synthase-encoding gene and one uroporphyrinogen III methyltransferase-encoding gene was still upregulated (Fig. 4B, Supplementary Table S12), suggesting that these three genes might play a crucial role in light energy absorption and photosynthetic electron transport in P. cornutum under saline conditions. Furthermore, the majority of the upregulated DEGs involved in carbon fixation after 6 h of salt treatment were also upregulated after 24 h of salt treatment (Fig. 4, Supplementary Table S11 and S12).

DEGs related to transcription factors

Along with functional genes, regulatory genes also participate in the responses of plants to environmental stresses by regulating signal transduction or functional gene expression [40]. Transcription factors (TFs) are important and abundant regulatory genes in higher plants. The major classes of TFs include NAC (NAM/ATAF/CUC), AP2/ERF (APETALA2 and ethylene-responsive element binding proteins), bHLH (basic helix-loop-helix), MYB (myeloblastosis), WRKY (WRKY-domain), bZIP/HD-ZIP (basic region-leucine zipper/homeodomain-leucine zipper), ZF (zinc finger) and HSP (heat shock protein), some of which have been validated to confer salt and drought tolerance in various plant species by transcriptional regulation of downstream target stress-responsive genes [9]. As TFs generally show rapid responses to abiotic stresses, we only analyzed the differentially expressed TF genes in roots and shoots after 6 h of salt treatment (the number of the differentially expressed TF genes after salt treatment for 24 h was much lower than that after salt treatment for 6 h in our results; data not shown).

After 6 h of 50 mM NaCl treatment, more than 100 TF genes were differentially expressed in roots; approximately two-thirds were upregulated, among which the majority were WRKYs, MYBs, ZFs, or bZIPs/HD-ZIPs, including WRKY33, WRKY54, and MYB3, CCCH-type ZFs and HD-ZIP6 (Fig. 5A, Supplementary Table S13), which have been validated to play important roles in salt tolerance and drought resistance in other plants [41–45]. Thus, the expression of these TF genes in roots should also be essential for salt tolerance of P. cornutum. Although less research have shown that MADS-box genes are closely related to the responses of plants to salinity, all 5 differentially expressed MADS-box genes in roots after salt treatment were considerably upregulated, with 3 members (AGL16, AGL27 and AGL29) showing no expression under the control condition but high expression under salt treatment (Fig. 5A, Supplementary Table S13). Up to 6 upregulated AP2/ERF-encoding genes were also found in roots. Only a few NAC, bHLH and HSP-encoding genes were differentially expressed after salt treatment (Fig. 5A).

After 6 h of 50 mM NaCl treatment, nearly 120 TF genes were differentially expressed in shoots (Fig. 5B). The number of upregulated WRKY and AP2/ERF-encoding genes in shoots was much lower than that in roots (Fig. 5), suggesting that WRKYs and AP2/ERFs might mainly regulate stress-responsible functional genes in roots to confer salt tolerance in P. cornutum. Similarly, upregulated MADS-box-encoding genes in shoots were less abundant than in roots after salt treatment, but the expression of MADS-box gene AGL30 was upregulated more than 5-fold in both shoots and roots (Fig. 5, Supplementary Table S13 and S14), suggesting that this TF is distinctively implicated in salt tolerance in P. cornutum. Many upregulated MYBs, including six members that showed no expression under the control condition, and ZFs, including seven members that showed no expression under the control condition, were also observed in shoots after salt treatment (Fig. 5B, Supplementary Table S14). Notably, upregulated HSP-encoding genes were only found in shoots but not in roots (Fig. 5), suggesting that HSPs might mainly regulate stress-responsible functional genes in shoots of P. cornutum under saline conditions.

Real-time PCR validation of differential gene expression

A real-time PCR validation was performed using 40 DEGs (20 randomly selected from roots and other 20 randomly selected from shoots). As shown in Supplementary Fig. S2, the fold changes of the selected DEGs measured by qRT-PCR were highly consistent with the results obtained from RNA-seq data. The correlation coefficient R² of the DEGs from roots between qRT-PCR and RNA-seq results under salt treatment for 6 and 24 h was 0.93 and 0.92 respectively (Supplementary Fig. S2A and B). The correlation coefficient R² of the DEGs from shoots between qRT-PCR and RNA-seq results under salt treatment for 6 and 24 h was 0.86 and 0.91 respectively (Supplementary Fig. S2C and D). Hence, the RNA-Seq results in the present study were highly reliable.

Ion transport plays a vital role in the response to salt stressin P. cornutum

Cl^- is the most common and abundant anionic salt in saline soils [1]. For most crops, especially legumes and perennial woody species, Cl^- toxicity is a major cause of yield reduction under saline conditions [21, 46]. However, it has been proven that the large absorption of Cl^- from external surroundings and translocation of Cl^- from roots into shoots for osmotic adjustment are important physiological mechanisms in salt tolerance of the xerophyte P. cornutum, which can adapt well to halomorphic arid soils [19, 20]. Therefore, P. cornutum must have evolved multiple molecular mechanisms of Cl^- transport to survive in these harsh conditions.

Until recently, the molecular mechanisms underpinning Cl^- transport in higher plants were poorly defined and the molecular identities of certain basic Cl^- transport processes remained elusive [21]. As a micronutrient, Cl^- is primarily absorbed at the epidermis cells of root hairs via several transporters and channels [31]. Decades ago, active Cl^- transporters such as Cl^-/2H⁺ symporters and passive Cl^- influx/efflux channels at the plasma membrane of root epidermis were identified using electrophysiological approaches [47, 48], whereas to date, the corresponding candidate genes are still unknown. A previous work on Zea mays proposed a hypothesis that ZmNPF6.4 is likely a component of the plant root Cl^- uptake system, as ZmNPF6.4 is a plasma membrane-localized, proton-coupled, chloride-selective transporter in root epidermis cells [49]. However, the transcriptional responses of NPF6.4 in plants to salt stress has not been investigated. In the present study, two transcripts, NPF6.4a (CL7387.Contig1_All) and NPF6.4b (CL7387.Contig2_All), for NPF6.4 in roots of P. cornutum were found. Although the transcript abundance of NPF6.4b in roots was repressed by NaCl treatment for 24 h, the transcript abundance of NPF6.4a was continuously induced by NaCl treatment for both 6 and 24 h (Supplementary Table S3 and S4), indicating that NPF6.4ais very likely to facilitate the uptake of Cl^- by roots in P. cornutum under saline conditions.

The overaccumulation of Cl^- in shoots generally triggers Cl^- toxicity in photosynthetic organs and ultimately restricts plant growth [1, 50]. The key rate-limiting gatekeeper step modulating Cl^- accumulation in the shoots has been shown to be the loading of Cl^- from the root stelar symplast into the xylem apoplast [21]. AtSLAH1 currently is the only known protein certainly mediating Cl^- loading into xylem under saline conditions [51, 52]. As A. thaliana is a Cl^--sensitive species, AtSLAH1 mediates excessive Cl^- translocated into shoots in this species; therefore, AtSLAH1 negatively regulates salt tolerance in A. thaliana. Indeed, the expression of AtSLAH1 in roots is substantially downregulated under salt stress [52], indicating that Cl^--sensitive species mainly avoid Cl^- overaccumulation by repressing the expression of SLAH1 in roots. Conversely, in the roots of P. cornutum, SLAH1 showed no expression under the normal condition but was highly expressed under salt treatment for both 6 and 24 h (Supplementary Table S3 and S4). Therefore, P. cornutum likely mainly accelerates its Cl^--accumulating characteristic by activating the expression of SLAH1 in roots. Therefore, the function of SLAH1 in regulating salt tolerance between salt-sensitive and -tolerant plant species should be distinctly different. In O. sativa, CCC1 has been shown to mediate Cl^- retrieval from xylem sap into roots, and the expression of OsCCC1 in roots is increased under salt stresses to reduce the Cl^- accumulation in shoots [23]. Our results showed that the transcript abundance of CCC1 in roots of P. cornutum was downregulated after salt treatment (Fig. 2B), which should contribute to decreasing the amount of Cl^- withheld in roots and, ultimately, help to translocate more Cl^- into shoots. Therefore, CCC1 is likely also an important component in regulating Cl^- accumulation of P. cornutum under saline conditions.

AtCLCg is a unique protein mediating Cl^- compartmentalization into vacuoles of mesophyll cells [33]. In the typical Cl^--sensitive plant Glycine max, GmCLC1, a homolog of AtCLCg, is preferentially expressed at the tonoplast of root cells; therefore, the expression of GmCLC1 contributes to withholding more Cl^- in roots to decrease Cl^- accumulation in shoots [53]. In the present study, the transcript abundance of P. cornutum CLCg in shoots was also induced by salt treatment for both 6 and 24 h (Table 3). Considering that P. cornutum is a typical Cl^--accumulating plant, the expression of CLCg in shoots under salt treatment might be essential for decreasing Cl^- toxicity to mesophyll cells and synergistically helping to store more Cl^- in shoots for enhancing osmotic adjustment capacity. Therefore, there should be a distinctly different responsive model of CLCg to salt stress between Cl^--sensitive and -tolerant species.

The uptake and storage of NO₃^- in plants under salt conditions is generally antagonized by the uptake of Cl^-, which competes with NO₃^- for the major binding sites of transmembrane channels or transporters [21, 48]. Our previous studies have shown that the capacity to maintain NO₃^-homeostasis in shoots is also a principal trait in the Cl^--accumulating capabilityof P. cornutum under saline conditions [19, 20]. The NO₃^- uptake by plant roots is mediated by the nitrate transporter NPF6.3 (also known as CHL1 or NRT1.1) [54]. Our results found two transcripts, NPF6.3a (CL8124.Contig1_All) and NPF6.3b (CL8124.Contig3_All), for NPF6.3 in roots of P. cornutum, and the former was upregulated after 24 h of salt treatment, the latter was upregulated after 6 h of salt treatment (Supplementary Table S3 and S4), which would synergistically activate a strong NO₃^- uptake capacity at the root epidermis. Hence, NPF6.3 likely also plays an essential role in salt tolerance in P. cornutum. Root-to-shoot transport of NO₃^- in plants is mediated by NPF7.3 (also known as NRT1.5) [55]. In A. thaliana, the expression of AtNRT1.5 in roots is downregulated in response to NaCl treatment [56]. However, in the present work, P. cornutum NPF7.3 was not among the DEGs after salt treatment, indicating that the expression of NRT1.5 could remain unchanged to render a constant NO₃^- transport from roots into shoots under salt treatment. Therefore, the superior long-distance transport ability of NO₃^- regulated by NRT1.5 likely represents another important trait in the salt tolerance of P. cornutum.

Efficiency in maintaining Na⁺ and K⁺ homeostasis is crucial for salt tolerance in higher plants [1]. Numerous proteins involved in these processes have been identified and functionally characterized. For example, NCX and CNGC mediate root Na⁺ and/or K⁺ uptake [57, 58], HKT1 and SOS1 mediate Na⁺ long-distance transport from roots to shoots [25, 36], KT/KUP/HAK mediate K⁺ transport in roots [59, 60], and tonoplast NHX mediates Na⁺ and/or K⁺ compartmentalization into vacuoles [24]. In the present study, we also found that many genes encoding the abovementioned proteins were upregulated after salt treatment (Fig. 2), suggesting that in P. cornutum, these genes also primarily regulate Na⁺/K⁺ homeostasis. It has been proven that Zygophyllum xanthoxylum, another important xerophyte in northwestern China, could accumulate extremely high Na⁺ concentrations in leaves as an important osmoticum to improve photosynthesis and hydration under salt and drought stresses [61, 62]. Subsequent studies have validated the vital roles of many Na⁺ and/or K⁺ transporters/channels in maintaining Na⁺/K⁺ homeostasis in Z. xanthoxylum [63, 64]. Therefore, the molecular mechanisms underlying Na⁺ and K⁺ transport in xerophytic desert plants are much clearer than those involved in Cl^- transport. However, our results provide new insights into Na⁺/K⁺ homeostasis in desert plants. For example, the transcript abundance of HKT1 in P. cornutum was upregulated in shoots but remained stable in roots under salt treatment for both 6 and 24 h (Fig. 2), indicating that HKT1 is mainly responsive to salt in shoots of P. cornutum. To date, the molecular factors involved in Na⁺ uptake into mesophyll cells in plants are still unknown. As HKT1-type proteins locate at the plasma membrane and mediate Na⁺ transport [36, 37], we can hypothesize that the expression of HKT1 in shoots might contribute to Na⁺ entry into shoots to use Na⁺ as an osmoticum in P. cornutum. Furthermore, the transcript abundances of NHX5 and NHX6 were upregulated in roots and shoots of P. cornutum under salt treatment for both 6 and 24 h (Table 2 and 3). In A. thaliana, both NHX5 and NHX6 are Golgi and trans-Golgi network-located Na⁺/H⁺ antiporters that synergistically trigger plant salt tolerance by maintaining organelle pH and ion homeostasis [65]. However, the function of NHX5 and NHX6 in salt tolerance of desert plants has not been investigated. Our results suggested that both NHX5 and NHX6 might also play essential roles in that adaptation of desert plants to harsh environments.

The ROS-scavenging system are important for salt stress adaptation in P. cornutum

Plants undergo a series of oxygen metabolism reactions during growth, causing them to accumulate a certain amount of ROS in the cells. Under salt stress, the destruction of intracellular osmotic homeostasis can lead to dysfunction in ROS generation and scavenging mechanisms, resulting in the considerable accumulation of oxygen free radicals in the cells, which in turn exert toxic effects on the cells [66]. Previous works have shown that under salt and drought stresses, P. cornutum exhibits a high ROS-scavenging capacity, with markedly increased SOD, CAT and POD activities [16–18].

SOD is the primary antioxidant enzyme in the ROS-scavenging system. It can convert superoxide anion radicals into free oxygen radicals and hydrogen peroxide. Subsequently, the large amounts of resulting free oxygen radicals and hydrogen peroxide are scavenged mainly through the ASA-GSH cycle, the GPX pathway, the CAT pathway, and the PrxR/Trx pathway [67]. SODs in higher plants are classified by their metal cofactors into three known types: the Cu/Zn SOD, Mn SOD and Fe SOD, among which Cu/Zn SOD has the widest subcellular distribution (cytosolic fraction and chloroplast) [66]. The upregulation of SODs has been proven to substantially contribute to combating oxidative stress resulting from both biotic and abiotic stresses [68, 69]. In this study, the transcript abundance of Cu-Zn SOD was upregulated after salt treatment for 24 h in both roots and shoots of P. cornutum (Fig. 3, Supplementary Table S8 and S10). Hence, Cu-Zn SODshould be a core component in the ROS-scavenging system of P. cornutum confronted with soil salinity. CAT is indispensable for ROS detoxification during stress conditions; it has the highest efficiency for converting H₂O₂ in plant cells into H₂O and O₂ among all the antioxidant enzymes in plants [70]. In the present study, only the CAT3 was induced in roots and shoots by salt treatment (Fig. 3, Supplementary Table S8 and S10), suggesting that under saline conditions, P. cornutum mainly increases its CAT activity by upregulating the expression of CAT3 in roots and shoots. Among the upregulated DEGs involved in the four ROS-scavenging pathways in roots and shoots after salt treatment, most were GSTs and Trxs (Fig. 3). In the AsA-GSH cycle and GPX pathway, glutathione (GSH) acts as a sensor of redox to maintain lower levels of ROS; it must be first catalyzed by GSTs as an antioxidant, after which it can function in ROS scavenging [67]. Many studies have found that the overexpression of GSTs could substantially improve ROS-scavenging capacity in plants under salt stress [71, 72]. Therefore, the upregulated GSTs identified in the current study likely also play vital roles in ROS-scavenging system of P. cornutum under saline conditions (Fig. 3). The thioredoxin (Trx) proteins serve as redox transmitters within the cellular thiol/disulfide redox network, and the peroxiredoxin (PrxR) proteins act as thiol-dependent peroxidases with high affinity for peroxides, especially for H₂O₂, to protect protein thiols from oxidation. Thus, the PrxR/Trx pathways also play an important role in the ROS-scavenging system in plants [73]. In the present study, many Trx genes in roots and shoots were upregulated after salt treatment (Fig. 3), suggesting that Trx may also play essential roles in the ROS-scavenging system of P. cornutum under 50 mM NaCl treatment.

High efficiency for stomatal movement and carbon fixation is essential for the adaption of P. cornutum to salt stress

Stomatal closure to reduce water loss through transpiration is an important strategy by which P. cornutum tolerates salt stress, which in turn triggers stomatal limitation for photosynthesis; however, although the net photosynthetic rate of P. cornutum is significantly decreased, its dry biomass remains stable due to its high carbon assimilation capacity [19]. Therefore, the genes implicated in stomatal movement and carbon assimilation play essential roles in salt tolerance in P. cornutum.

The aperture of the stomatal pore is regulated by changes in the osmotic potentials of the guard cells. These changes are mainly achieved by transporting K⁺ and organic as well as inorganic anions across cellular membranes [74]. The outward K⁺ channels GORK participate in K⁺ efflux through the plasma membrane of guard cells and are consequently involved in stomatal movement [38]. In the present study, the salt-induced expression of GORK in shoots of P. cornutum was obvious (Fig. 2D), suggesting that these genes are closely related to stomatal closure in P. cornutum confronted with salt stress. In A. thaliana, several anion transporters also mediate NO₃^- and/or Cl^- transport across the plasma membrane or tonoplast of guard cells. For example, SLAC1 and ALMT12 facilitate NO₃^- and/or Cl^-release or uptake from guard cells, while CLCc and ALMT9 mediate Cl^- compartmentalization into vacuoles of guard cells [75, 76]. In P. cornutum, the transcript abundance of these genes in shoots was unaltered after salt treatment, indicating that there are other components involved in anion release or uptake from guard cells.

Photosynthetic electron transport is the primary step in the process of oxygenic photosynthesis, which converts sunlight into active chemical energy to provide ATP for the subsequent carbon assimilation [39]. In the present study, numerous upregulated genes associated with all the major components of photosynthetic electron transport, such as chlorophyll biosynthesis, the PS II complex, the PS I complex, the cytochrome b₆f complex, the ATP synthase and ferredoxin, were identified in the shoots of P. cornutum after salt treatment for either 6 or 24 h (Fig. 4). Therefore, these photosynthetic electron transport-related genes in shoots of P. cornutum are very likely to be involved in the enhancement of carbon assimilation by activating a stronger capacity for light energy conversion and ATP generation. Furthermore, our results identified many upregulated genes that encode various enzymes directly involved in carbon assimilation, including phosphoenolpyruvate, malate dehydrogenase, alanine aminotransferase/transaminase, ribulose–1,5-diphosphate carboxylase, phosphoglycerate kinase and glyceraldehyde–3-phosphate dehydrogenase-encoding genes (Fig 4, Supplementary Table S11 and S12). Hence, these genes likely play central roles in enhancing the carbon assimilation capacity of P. cornutum under saline conditions.

Transcription factors play important roles in regulating salt-responsive genes in P. cornutum under salt stress.

Studies of salt- and drought-signaling networks unequivocally support a key and integrative function of members of the bZIP TFs in these regulatory networks, and the potential of these factors to confer enhanced stress tolerance has been demonstrated repeatedly [77]. A key regulator of salt stress adaption is bZIP24, which confers increased salt tolerance to A. thaliana by stimulating the transcription of a wide range of stress-inducible functional genes such as HKT1 and SOS1 [78]. In the present study, the transcript level of bZIP24 in roots of P. cornutum was upregulated after salt treatment (Supplementary Table S13), suggesting that the expression of bZIP24 in roots might predominately regulate transcriptional networks in salt stress adaption of P. cornutum. WRKY proteins regulate diverse plant processes including biotic and abiotic stresses adaption [79]. In A. thaliana, involvement of WRKY factors in plant salt adaptation was shown for WRKY25 and WRKY33 [41]. In our study, the WRKY33 transcript WRKY33a was highly induced by salt in both roots and shoots of P. cornutum, and another transcript, WRKY33b, was induced by salt in roots (Supplementary Table S13 and S14). Therefore, WRKY33likely also plays essential roles in salt signaling networks in P. cornutum. The Cys2/His2-type ZF proteins have been proven to control and regulate WRKY functions, the ROS-signaling pathway and stomatal closure [80, 81]. In our results, two transcripts (CL7040.Contig1_All and CL7040.Contig2_All) for Cys2/His2 2 were both induced by salt treatment in shoots (Supplementary Table S14), indicating that these two genes might also be important for salt tolerance in P. cornutum by regulating multiple processes such as WRKY function, ROS scavenging and stomatal movement. In addition to Cys2/His2-type ZF proteins, certain members of the bHLH family, such as bHLH92, confer salt tolerance in plants also by controlling ROS-scavenging signaling as various PODs are their downstream targets [82]. In P. cornutum, the transcript abundance of bHLH92 in shoots was upregulated after salt treatment (Supplementary Table S14). Thus, bHLH92 likely participates in the enhancement of ROS-scavenging capacity in salt adaption of P. cornutum. The members of the MADS-box TF family were previously thought to be mainly involved in organ development such as floral emergence and root architecture [83, 84]. However, in the present study, some MADS-box genes showed almost no expression under the normal condition but were highly expressed in shoots or roots of P. cornutum under saline conditions (Supplementary Table S13 and S14). Thus, we can hypothesize that MADS-box TFs might play specific roles in regulating salt tolerance in desert plant species. Additionally, our results identified other salt-inducible TF genes in P. cornutum, such as NAC62 and MYB59 (Supplementary Table S13 and S14), whose regulatory mechanisms in salt adaption in other plants have been less intensively investigated. Thus, these genes might represent novel regulators in salt tolerance in P. cornutum.

This study provides a first analysis of gene transcripts in P. cornutum under and salt treatments. Candidate genes that probably confer salt tolerance of P. cornutum by facilitating Cl^- accumulation and NO₃^- hemostasis, as well as the transport of other inorganic osmoticums such as Na⁺, K⁺, SO₄^2- and PO₄^3- were identified. Meanwhile, many salt-responsive genes associated with the enhancement of ROS-scavenging capacity and carbon assimilation efficiency to improve the salt tolerance of P. cornutum were identified. Additionally, possible transcription factor genes with multiple functions in regulating the adaption of P. cornutum to salt stress were found. These results would promote the research on the molecular mechanisms of salt tolerance in the xerophytic species and lay a foundation for genetic improvement of stress-resistance of important forage and crop species in arid areas

Plant growth and treatments

All the species of the genus Pugionium have been identified by many researchers according to differences in silicle wings and the lobe shape of the cauline and radical leaves [13, 85, 86]. P. cornutum is the model species of this genus, this species is a wild vegetable in desert area of northwestern China, and it is free to collect this plant and its seeds [14, 15]. In the present study, seeds of P. cornutum were collected from plants grown in Mu Us Sandland in the Inner Mongolia Autonomous Region, China. Corresponding voucher specimen (No. Q. S. Yu, 6065) has been deposited in the Herbarium of School of Life Science, Lanzhou University [13]. We identified these collected seeds according to their typical features described by Yu et al. [13]. After removal of the bracts, seeds were disinfected with 75% alcohol for 30 s, sterilized with 5% NaClO for 10 min, and then rinsed 6 times with distilled water, soaked in distilled water for 1 day and then germinated at 28°C in the dark. After approximately 5 days, the seedlings were transplanted to plastic pots (one seedling/pot) filled with coarse silica sand and irrigated with Hoagland solution containing 2 mM KNO₃, 0.5 mM KH₂PO₄, 0.5 mM MgSO₄, 0.5 mM Ca(NO₃)₂, 60 µM Fe-citrate, 50 µM H₃BO₃, 10 µM MnCl₂, 1.6 µM ZnSO₄, 0.6 µM CuSO₄, and 0.05 µM Na₂MoO₄ (pH was adjusted to 5.7 by using 1 M Tris). The solution was refreshed every 3 days. All seedlings were grown in a greenhouse, where the daily photoperiod was 16 h/8 h (light/dark), the temperature was 28°C/23°C (day/night), the light flux density was approximately 500 µmol m^–2 s^–1 and the relative humidity was approximately 60%.

After one month, uniform seedlings were randomly divided into two groups: control group (C) and salt group (S). In the control group, seedlings were irrigated with normal Hoagland solution; after 6 and 24 h, root (R) and shoot (S) samples were collected and labeled as C6R, C24R, C6S, and C24S, respectively. In the salt group, seedlings were treated with Hoagland solution containing 50 mM NaCl; after 6 and 24 h, root (R) and shoot (S) samples were collected and labeled as S6R, S24R, S6S, and S24S, respectively. Each sample was collected from 5–6 individual seedlings, and immediately frozen in liquid nitrogen for total RNA extraction.

Transcriptome sequencing

Total RNA was extracted from the four root samples (C6R, C24R, S6R, S24R) and four shoot samples (C6S, C24S, S6S, S24S) using the RNeasy Plant Mini Kit (Qiagen, Netherlands). Then, residual DNA was removed from the extracted RNA by treatment with DNase I at 37°C for 40 min. RNA integrity and purity were determined using a NanoDrop ND 1000 instrument (Thermo Scientific).

To obtain the transcriptome in roots and shoots of P. cornutum, the four RNA samples from roots were pooled together and the four RNA samples from shoots were pooled together. Poly (A) mRNA in the two pooled total RNA samples was purified and then fragmented into small pieces as described by Qiu et al. [87]. The first-strand cDNA and second-strand cDNA of the mRNA fragments were synthesized for end-repair and poly (A) addition and then linked to sequencing adapters according to Dang et al. [88]. Finally, we constructed two paired-end cDNA libraries (one for root and one for shoot) and sequenced the cDNA in each library on an Illumina HiSeq™ 2000 platform at the Beijing Genomics Institute (BGI, Shenzhen, China).

The raw sequencing reads were filtered to obtain high-quality clean reads as described by Qiu et al. [87], and then transcriptome de novo assembly and unigene acquisition were performed [12, 88]. Finally, the obtained unigenes were clustered into the following two classes: (i) clusters: each cluster contained several unigenes sharing ≥ 70% sequence similarity with each other that were named with a CL + cluster digital ID as the prefix followed by the contig number (e.g., CL1. Contig 1, 2, 3…); (ii) singletons: each singleton represented one individual sequence that did not reach 70% similarity with any other sequence or fall into any assembly and was named with Unigene as the prefix followed by the gene digital ID (e.g., Unigene 1, 2, 3…). To obtain their protein functional annotations, the all-unigene sequences were aligned against the protein databases, including Nr, Nt, Swiss-Prot, KEGG, COG and GO, using BLASTX with a significance threshold of E-value < 10^–5.

Differentially expressed genes (DEGs) analysis

The eight extracted total RNA from P. cornutum tissue samples were used to construct eight independent sequencing tag libraries (C6R, C24R, S6R, S24R, C6S, C24S, S6S and S24S) in parallel with a tag-based digital gene expression (DGE) system according to Ma et al. [12] and Xue et al. [89]. Then, each sequencing tag library was sequenced on an Illumina HiSeq™ 2000 platform at the Beijing Genomics Institute.

The obtained tags in each library were filtered as described by Ma et al. [12]. Then, the clean tags in each cDNA library were mapped to the all-unigenes data from the transcriptome sequencing using the short reads alignment program SOAPaligner/soap2 to determine the protein functional annotations of the genes. The transcript abundance of each gene was determined by the RPKM (reads per kilobase of exon model per million mapped read) method [90].

To identify genes responding to salt treatment, the log₂ratio of gene transcript abundance between the salt-treated tissue sequencing tag library and the corresponding untreated tissue sequencing tag library (S6R vs. C6R, S24R vs. C24R, S6S vs. C6S, S24S vs. C24S) was calculated. The statistical significance of the differential expression value for each gene was determined by evaluating the probability (P-value) of a gene between two tag libraries being transcribed equally using the formula proposed by Audic et al. [91]. The results of all statistical tests were corrected for multiple tests, and the false discovery rate (FDR) was used to determine the threshold P-value in multiple tests [24]. In this study, a gene with FDR < 0.001 and the absolute value of log₂ratio > 1 was termed as a differentially expressed gene (DEG).

Real-time quantitative PCR validation of DEG results

Independent real-time quantitative PCR (qPCR) was conducted to validate the DEG results from RNA sequencing. P. cornutum seedlings were cultured and treated exactly the same as described above. We randomly selected 20 DEGs from roots and 20 DEGs from shoots under both 6 and 24 h of salt treatments from our RNA sequencing data. Then, the expression changes of these DEGs under 50 mM NaCl treatment relative to the control condition were investigated using the qPCR method as described by Yuan et al. [63], where ACTIN2 served as an internal reference. Subsequently, a correlation analysis of the obtained data between RNA sequencing and qPCR was performed.

RPKM: reads per kilobase of exon model per million mapped read; DEG: Differentially expressed gene; FDR: false discovery rate; SLAH: slow type anion channel; CCC1: cation-chloride co-transporter 1; CLC: chloride channel; NPF: nitrate transporter 1/peptide transporter; SOS1: plasma membrane Na+/H+ antiporter; NHX: tonoplast Na+/H+ antiporter; NCX: Na⁺/Ca²⁺ exchanger; GLR: glutamate receptor; HKT: high-affinity K+ transporter; KT/HAK/KUP: K+ transporter; KEA: K⁺ efflux antiporter; SKOR: stelar K+ outward rectifying channel; CNGC: cyclic nucleotide-gated channel; V-CHX: vacuolar cation/H⁺ exchanger; GORK: guard cell outward-rectifying K⁺ channel; Sultr: SO42− transporter; PhT: PO43− transporter; ZnT: Zn2+ transporter; CTR: Cu2+ transporter; MnT: Mn²⁺ transporter; MGT: Mg2+ transporter; AMT: NH4+ transporter; IRT: iron transporter; BOR: BO₃3− transporter; V-H+ ATPase: vacuolar H+ ATPase; P-H+ ATPase: plasma membrane H+ ATPase; P-Ca2+ ATPase: plasma membrane Ca2+ ATPase; ROS: reactive oxygen species; GLR: glutaredoxin; APX: ascorbate peroxidase; GST: glutathione S-transferase; GPX: glutathione peroxidase; POD: peroxidases; CAT: catalase; PEX: peroxisomal biogenesis factor; Trx: thioredoxin; PrxR: peroxiredoxin; SOD: superoxide dismutase; HSP: heat shock transcription factor; ZF: zinc finger protein; MADS-box: MADS-box transcription factor; bHLH: basic helix-loop-helix protein; NAC: NAM/ATAF/CUC; AP2/ERF: APETALA2 and ethylene-responsive element binding proteins; bZIP/HD-ZIP: basic leucine zipper/ homeodomain-leucine zipper protein; MYB: MYB-domain protein; WRKY: WRKY-domain protein

Acknowledgements

Not applicable.

Funding

This work was financially supported by the National Natural Science Foundation of China (Grant No. 31730093 and 31501994), the National Key Research and Development Program of China (grant No. 2017YFC0504804), the Fundamental Research Funds for the Central Universities (lzujbky–2019–40 and lzujbky–2018-it15). Funds were used for the design of the study, collection, analysis, and interpretation of data and in writing the manuscript, as well as in the open access payment.

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Authors’ contributions

QM designed the study, Y-NC performed the experiment, Y-NC, F-ZW, C-HY, J-ZY, HG collected and analyzed the data, Y-NC wrote the manuscript, QM, J-LZ, S-MW revised the manuscript. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Table 1 Summary of de novo sequence assembly

Unigenes	Total number	Total length (bp)	Mean length (bp)
Shoot	64978	70865211	1091
Root	80307	80091654	997
All	72086	89575317	1243

Table 2 The upregulated DEGs related to ion transport in roots of P. cornutum after 50 mM NaCl treatment for both 6 and 24 h.

Gene ID	Protein	Function
CL5647.Contig1_All	SLAH1	Cl^- transport from roots to shoots
CL2477.Contig4_All	CLCg	Cl^- vacuole compartmentalization
CL7387.Contig1_All	NPF6.4	Cl^- uptake at root epidermis
CL1096.Contig4_All	NHX5	Na⁺/H⁺ antiporter
CL1096.Contig10_All	NHX6	Na⁺/H⁺ antiporter
CL3604.Contig8_All	GLR3.3	Cation channel activator
CL3206.Contig3_All	Sultr1.2	SO₄^2- uptake
CL7785.Contig1_All	Sultr3.3	SO₄^2- transporter
CL564.Contig8_All	PHT4.3	PO₄^3- transporter
CL2740.Contig1_All	PDR2	Mn²⁺ transporter
CL666.Contig1_All	BOR4	BO₃^3- exporting from cytoplasm

Table 3 The upregulated DEGs related to ion transport in shoots of P. cornutum after 50 mM NaCl treatment for both 6 and 24 h.

Gene ID	Protein	Function
CL4002.Contig2_All	CCC1	Cl^- retrieval from xylem sap
CL2577.Contig2_All	CLCf	Cl^- channel
CL2477.Contig4_All	CLCg	Cl^- vacuole compartmentalization
Unigene7084_All	NPF5.2	Cl^- and/or NO₃^- transporter
CL3282.Contig2_All	NPF6.2	Cl^- and/or NO₃^- transporter
CL6278.Contig1_All	NPF8.1	Cl^- and/or NO₃^- transporter
CL2487.Contig3_All	NPF5.9	Cl^- and/or NO₃^- transporter
CL1096.Contig4_All	NHX5	Na⁺/H⁺ antiporter
CL1096.Contig10_All	NHX6	Na⁺/H⁺ antiporter
CL3834.Contig2_All	HKT1	Na⁺ and/or K⁺ cellular efflux
CL1280.Contig5_All	KUP5	K⁺ transporter
Unigene6900_All	CNGC1	Ca²⁺ root uptake
Unigene13264_All	CTR5	Cu²⁺ transporter
Unigene1617_All	MGT10	Mg²⁺ transporter

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Transcriptomic profiling identifies candidate genes involved in salt tolerance of the xerophyte Pugionium cornutum

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