Physiological and transcriptomic analyses reveal the mechanisms underlying the salt tolerance ofZoysia japonicaSteud

Background

地区with saline soils are sparsely populated and have fragile ecosystems, which severely restricts the sustainable development of local economies.Zoysiagrasses are recognized as excellent warm-season turfgrasses worldwide, with high salt tolerance and superior growth in saline-alkali soils. However, the mechanism underlying the salt tolerance ofZoysiaspecies remains unknown.

Results

The phenotypic and physiological responses of two contrasting materials,Zoysia japonicaSteud. Z004 (salt sensitive) and Z011 (salt tolerant) in response to salt stress were studied. The results show that Z011 was more salt tolerant than was Z004, with the former presenting greater K⁺/Na⁺ratios in both its leaves and roots. To study the molecular mechanisms underlying salt tolerance further, we compared the transcriptomes of the two materials at different time points (0 h, 1 h, 24 h, and 72 h) and from different tissues (leaves and roots) under salt treatment. The 24-h time point and the roots might make significant contributions to the salt tolerance. Moreover, GO and KEGG analyses of different comparisons revealed that the key DEGs participating in the salt-stress response belonged to the hormone pathway, various TF families and theDUFfamily.

Conclusions

Zoysiasalt treatment transcriptome shows the 24-h and roots may make significant contributions to the salt tolerance. The auxin signal transduction family, ABA signal transduction family,WRKYTF family andbHLHTF family may be the most important families inZoysiasalt-stress regulation.

土壤盐渍化是一个世界性的问题。地区with saline soils are sparsely populated and have fragile ecosystems, which severely restricts the sustainable development of local economies. As an important part of landscaping, turf plays an important role in protecting, improving and beautifying urban environments. Therefore, it is particularly important to choose high-quality salt-tolerant turfgrass suitable for landscaping in areas with saline soils.ZoysiaWilld。属的多年生植物属于吗the familyPoaceae, subfamilyChloridoideae, tribeZoysieae[51]。Zoysiagrasses are recognized as excellent warm-season turfgrasses worldwide; they are with salt tolerant, hardy, and drought tolerant and are widely used in athletic fields, home lawns and parks [10]。Compared with otherPoaceaefamily members,Zoysiagrasses have received less attention in the research community. However, as an alternative grass species for landscaping in saline-alkali soil,Zoysiahas superior growth qualities [26]。In particular, among the three most important commercial species,Zoysia japonicaSteud. is distinctly tolerant to abiotic stress [51]。Therefore, studying the salt tolerance ofZoysiaplants is highly important.

Previous studies on salt tolerance ofZoysiamainly focused on the evaluation of salt tolerance and the physiological mechanisms governing salt tolerance. Salt tolerance evaluations have shown that the salt tolerance ofZoysiaplants has rich genetic variation [25,35,40,57]。This variation makes for convenient selection of materials with contrasting salt tolerances for studying the salt tolerance mechanism ofZoysia.Zoysiaplants secrete salt; allZoysiaplant leaves have salt glands that regulate ion balance by selectively secreting salt ions. The salt tolerance ofZoysiaplants is positively correlated with the rate of Na⁺secretion from salt glands in leaves and the density of salt glands per unit leaf area [21,22,33]。Moreover, previous studies have shown that the salt tolerance ofZoysiais negatively correlated with the content of Na⁺and positively correlated with the content of K⁺in the leaf fluid. Salt-tolerant materials have a strong ability to maintain the K⁺/Na⁺ratio in their leaves and roots. The Na⁺content in leaves has been successfully used to evaluate the salt tolerance ofZoysia[25,33,34]。

The salt tolerance ofZoysiais a very important trait, but to date, its molecular regulatory mechanism remains unknown. The Na⁺/H⁺antiporter geneZjNHX1, which belongs to the plant NHX-gene family, was cloned fromZ. japonica, and studies have shown thatZjNHX1plays an important role in ion homeostasis and salt tolerance [9]。In addition, the glycine-rich RNA-binding protein-coding geneZjGRPwas isolated fromZ. japonicaand was strongly induced by NaCl treatment.ZjGRP-overexpressingArabidopsis thalianaplants present low germination rates, slow seedling growth and poor salt tolerance [50]。ZjZFN1is a C₂H₂-type zinc finger protein-coding gene that is expressed more in leaf tissues than in root and stem tissues, and its expression is induced by salt, cold and abscisic acid (ABA) treatments. OverexpressingZjZFN1inA. thalianacan improve seed germination and increase salt tolerance by improving the transcriptional activities of several salt-tolerance-related genes under salt stress [49]。

Studies on the salt tolerance genes ofZoysiaare scarce. However, using a full-length cDNA expression library in yeast, Chen et al. [5] systematically excavated the salt tolerance genes inZoysia matrellaand identified 16 candidate salt tolerance genes involved in ion regulation, osmotic adjustment, protein folding and modification, mitochondrial membrane translocase and RNA metabolism. Xie et al. [58] presented the first comprehensive transcriptome data ofZ. japonicaSteud. roots, and a total of 32,849 unigenes and 4842 simple sequence repeats (SSRs) were identified. Their results showed that transcription factors (TFs) including members of theAP2/EREBPfamily,bZIPfamily,NACfamily,WRKYfamily,MYBfamily andbHLHfamily play significant roles in the early response to salt stress [58]。

Studies of the salt tolerance of zoysiagrass so far have focused on evaluating the salt tolerance among different cultivars, the physiological mechanisms of salt tolerance and the development of molecular markers [11,59]。However, the molecular mechanism of salt tolerance in zoysiagrass remains unclear. In this study, we investigated the phenotypic and physiological responses of two materials with contrasting salt tolerances,Z. japonicaZ004 (salt sensitive) and Z011 (salt tolerant), in response to salt stress. On the basis of the existingZoysiareference genome [48], the HiSeq™ 2000 platform was used to perform RNA sequencing (RNA-seq) of the zoysiagrass leaves and roots. We then compared the transcriptomes at different time points (0 h, 1 h, 24 h, and 72 h) and of different tissues (leaves and roots) under salt treatments to identify the significant time points and tissues. According to the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of differentially expressed genes (DEGs) in different comparisons, the key DEGs participating in the salt-stress response were selected, and these DEGs belonged to the hormone pathway, TF families and theDUFfamily. Thus, our research provides fundamental information for use in future salt-stress studies ofZoysiaand improves the understanding of molecular mechanisms in salt-tolerant plants.

Phenotypic and physiological responses ofZ. japonicaSteud. To salt stress

Japanese lawngrass (Z. japonicaSteud.) is a popular and important warm-season turfgrass, and different accessions have different degrees of salt tolerance. In this study, two accessions with contrasting salt tolerances, Z004 (salt sensitive) and Z011 (salt tolerant), were chosen to analyse the salt tolerance mechanism ofZ. japonica. The salt treatment results showed that Z011 had strong salt tolerance and displayed good growth, while Z004 was sensitive to salt and withered and yellowed after treatment with 350 mM NaCl for 40 days (Fig.1a). Moreover, the leaf firing of Z004 was significantly greater than that of Z011 (Fig.1b), and the biomass statistics showed that the relative shoot clipping dry weight, verdure dry weight and root dry weight of Z011 were markedly greater than those of Z004 (Fig.1c-e).

To study the differences in the mechanism of salt tolerance between Z004 and Z011, the Na⁺and K⁺concentrations were measured in the leaves, roots and secretions. In the control (CK) group, the Na⁺concentrations and K⁺concentrations in the leaves, roots and secretions were not significantly different between Z004 and Z011 (Fig.2a-f). After treatment with 350 mM NaCl, the Na⁺concentrations in the leaves, roots and secretions of Z004 and Z011 were greater than those in the CK (Fig.2a-c). In the roots of Z004 and Z011 after NaCl treatment, the Na⁺concentrations were not different (Fig.2b). However, in the leaves, the Na⁺concentrations and secretions were significantly lower in Z011 than in Z004 (Fig.2a, c).

After treatment with 350 mM NaCl, the K⁺concentrations in the leaves of Z004 and Z011 were lower than those in the leaves of the CK, but there were no differences in the K⁺concentrations between Z004 and Z011 (Fig.2d). In addition, the K⁺concentrations in the roots of Z004 and Z011 were lower than those in the roots of the CK, and the K⁺concentration in Z011 was significantly greater than that in Z004 (Fig.2e). However, the K⁺secretion in Z004 and Z011 after treatment with NaCl was greater than that in the CK, and the K⁺secretion of Z004 was significantly greater than that of Z011 (Fig.2f). Comparing with Z004, Z011 maintained a greater K⁺/Na⁺ratio in both the leaves and roots (Fig.2g, h).

Transcriptome sequencing of the Z004 and Z011 accessions

Leaf and root samples for RNA-seq were collected at 0 h, 1 h, 24 h and 72 h after treating Z004 and Z011 with 350 mM NaCl. In total, 16 samples were sequenced on the HiSeq™ 2000 sequencing platform. We obtained an average of 28.8 million raw reads from the 16 libraries, and 97.18% of the sequences were confirmed as clean reads (Online Resource1). First of all, the total reads of our RNA-seq were mapped to the rice and sorghum genomes as references via Hisat2 (v2.0.5) software [17]。The results showed that the total reads to the rice reference genome (ftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/001/433/935/GCF_001433935.1_IRGSP-1.0) was 0.12–0.39% (Online Resource7), and the total reads to the sorghum reference genome (ftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/003/195/GCF_000003195.3_Sorghum_bicolor_NCBIv3) was 0.26–0.85% (Online Resource8). Afterward, the clean reads were mapped to the wholeZ. japonica基因组和62.7读取总数的-91.39%和61年。8–89.84% of the unique reads were mapped to the reference genome. Therefore, it is appropriate to select the zoysia genome as a reference. In addition, multiple-mapped reads constituted 0.85–1.67% of the total reads, and splice-mapped reads constituted 17.58–32.42% of the total reads. Furthermore, 73.44–77.17% of the total reads were mapped to exons in the reference genome, 13.38–16.10% of the reads were mapped to introns, and 8.58–12.06% of the reads were mapped to intergenic regions (Online Resource2). In total, 59,271 unigenes and 29,675 novel genes were revealed by the RNA-seq assays. There were more genes (62,172, 69.90%) with lengths exceeding 1000 bp than those (21,909, 24.63%) with lengths exceeding 300 bp and fewer than 1000 bp. For the convenience of comparison, we defined 0 h as the CK group and 1 h, 24 h and 72 h as the treatment (Tr) group. Principal component analysis (PCA) revealed that the leaf and root samples of Z004 and Z011 were separated into four different areas (Fig.3a). Among them, the Z011 root samples had the highest dispersion degree (Fig.3a). Moreover, the 24 h samples of the roots of Z004 and Z011 were separated from the other samples, indicating that 24 h could be a crucial time (Fig.3a).

Identification of DEGs in Z004 and Z011 in response to salt stress

To characterize the differences between Z004 and Z011 in response to salt stress, we explored the unigenes whose expression level significantly changed after NaCl treatment. The transcript abundance of each gene was calculated by their fragments per kilobase per million fragments (FPKM) values. edgeR software [41] was used to analyse the significant differences in expression with padj < 0.05 and fold change > 2, and DEGs were identified as having a |log2(fold change)| > 1 and padj < 0.05. Under salt stress conditions, a total of 4701 genes, 2591 genes, 4400 genes and 8846 genes were differentially expressed in the leaves of Z004 (Z0041L vs Z0040L, Z00424L vs Z0040L, Z00472L vs Z0040L), Z004 roots (Z0041R vs Z0040R, Z00424R vs Z0040R, Z00472R vs Z0040R), Z011 leaves (Z0111L vs Z0110L, Z01124L vs Z0110L, Z01172L vs Z0110L) and Z011 roots (Z0111R vs Z0110R, Z01124R vs Z0110R, Z01172R vs Z0110R), respectively. In addition, a total of 12,220 genes were differentially expressed in the leaf comparisons of Z011 and Z004 (Z0110L vs Z0040L, Z0111L vs Z0041L, Z01124L vs Z00424L, Z01172L vs Z00472L), and 22,439 genes were differentially expressed in the root comparisons of Z011 and Z004 (Z0110R vs Z0040R, Z0111R vs Z0041R, Z01124R vs Z00424R, Z01172R vs Z00472R) (Fig.3c). Interestingly, the number of DEGs of Z011 roots was obviously more than that in the Z004 roots, and the number of DEGs of roots was obviously more than leaves in comparisons of Z011 and Z004 (Fig.3b,c). These results indicated that the roots might make a significant contribution to the difference in salt tolerance between Z004 and Z011. Moreover, comparisons of the Z004 leaves, Z004 roots and Z011 leaves revealed more downregulated genes than upregulated ones (Fig.3b)。然而,在比较Z011根,re were more upregulated genes than downregulated ones (Fig.3b). In addition, the number of DEGs in the 24-h sample (Z01124R vs Z0110R) was 2.28 and 5.64 times that in the 1-h (Z0111R vs Z0110R) and 72-h (Z01172R vs Z0110R) samples. Venn diagrams were constructed that also show that, in the comparisons of the Z004 roots, Z011 leaves and Z011 roots, the number of specific DEGs was greater at 24 h than at 1 h and 72 h (Supplementary FigureS1). These results indicated that 24 h might be a relatively important time point for the salt-stress response.

GO analysis of DEGs in the Z004 and Z011 roots

For the functional characterization of DEGs, we assigned GO terms and selected significant GO classifications of DEGs in each comparison (padj < 0.05). Fourteen GO classes of ‘molecular function’ in the Z0041R vs Z0040R comparison were identified. In the comparison between Z00424R and Z0040R, 4 GO classes, 1 GO class and 20 GO classes fell into the categories ‘biological process’, ‘cellular component’ and ‘molecular function’ respectively. Meanwhile, in the Z00472R vs Z0040R comparison, the category ‘biological process’ had 2 GO classes, ‘cellular component’ had 1 GO class and ‘molecular function’ had 23 GO classes. In Z004R, 11 GO classes most relevant to the salt-stress response were concentrated in the Z00424R vs Z0040R comparison and Z00472R vs Z0040R comparison: ‘response to oxidative stress’, ‘response to stress’, ‘extracellular region’, ‘oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen’, ‘oxidoreductase activity, acting on peroxide as acceptor’, ‘antioxidant activity’, ‘peroxidase activity’, ‘iron ion binding’, ‘ubiquitin-protein transferase activity’, ‘ubiquitin-like protein transferase activity’ and ‘sequence-specific DNA binding’ (Online Resource3). These results showed that Z004 responded slowly to salt stress. A significant stress response did not occur after 1 h of NaCl treatment but did occur after 24 h and 72 h.

Six GO classes of ‘molecular function’ in the Z0041R vs Z0040R comparison were identified. In the comparison between Z00424R and Z0040R, 4 GO classes, 1 GO class and 20 GO classes fell into the categories ‘biological process’, ‘cellular component’ and ‘molecular function’ respectively. Meanwhile, in the Z00472R vs Z0040R comparison, the category ‘biological process’ had 2 GO classes, ‘cellular component’ had 1 GO class and ‘molecular function’ had 23 GO classes.

In the comparison between Z0111R and Z0110R, 6 GO classes, 16 GO classes and 10 GO classes fell into the category ‘biological process’, ‘cellular component’ and ‘molecular function’ respectively. In the comparison between Z01124R vs Z0110R, 10 and 5 GO classes fell into the category ‘biological process’ and ‘molecular function’ respectively. In the comparison between Z01172R vs Z0110R, 2 and 8 GO classes fell into the category ‘biological process’ and ‘molecular function’ respectively. In Z011R, 5 GO classes most relevant to the salt-stress response were identified within its three comparisons: ‘response to stress’, ‘response to oxidative stress’, ‘antioxidant activity’, ‘peroxidase activity’ and ‘oxidoreductase activity, acting on peroxide as acceptor’ (Online Resource3). These results showed that Z011 responded to salt stress faster than did Z004. The stress response occurred after 1 h of NaCl treatment. When the GO classes most relevant to salt stress in Z004R and Z011R were compared, it was found that the 5 GO classes in Z011R coincided with those in Z004R. However, ‘extracellular region’, ‘oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen’, ‘iron ion binding’, ‘ubiquitin-protein transferase activity’, ‘ubiquitin-like protein transferase activity’ and ‘sequence-specific DNA binding’ were unique to Z004R and might be related to the differences in salt tolerance between Z004 and Z011.

在Z0110R vs Z0040R比较,分类的biological process’ had 1 GO class and ‘molecular function’ had 8 GO classes. In the Z0111R vs Z0041R comparison, the category ‘biological process’ had 2 GO classes, ‘cellular component’ had 1 GO class and ‘molecular function’ had 22 GO classes. In the Z01124R vs Z00424R comparison, the category ‘biological process’ had 11 GO classes and ‘molecular function’ had 8 GO classes. In the Z01172R vs Z00472R comparison, the category ‘biological process’ had 2 GO classes and ‘molecular function’ had 8 GO classes (Online Resource3). Comparing with 0 h, the mutual GO class of Z011R vs Z004R comparisons among 1 h, 24 h and 72 h was ‘response to stress’. These results showed Z011 had faster and stronger stress response in salt tolerance.

KEGG pathway analysis of DEGs in the Z004 and Z011 roots

The DEGs in the Z004 and Z011 roots were mapped to KEGG pathways ofOryza sativa. In the Z0041R vs Z0040R, Z00424R vs Z0040R and Z00472R vs Z0040R comparisons, 40, 67 and 72 DEGs, respectively, involving 23, 40 and 32 pathways, respectively, were assigned to KEGG pathways (Online Resource4). In the Z0111R vs Z0110R, Z01124R vs Z0110R and Z01172R vs Z0110R comparisons, 100, 202 and 39 DEGs, respectively, involving 41, 52 and 26 pathways, respectively, were assigned to KEGG pathways (Online Resource4). In the Z0110R vs Z0040R, Z0111R vs Z0041R, Z01124R vs Z00424R and Z01172R vs Z00472R comparisons, 266, 114, 54 and 119 DEGs, respectively, involving 61, 44, 36 and 52 pathways, respectively, were assigned to KEGG pathways (Online Resource4). The major pathways identified in the above comparisons were ‘metabolic pathways’, ‘biosynthesis of secondary metabolites’, ‘plant hormone signal transduction’, ‘MAPK signalling pathway – plant’ and ‘phenylpropanoid biosynthesis’.

度的数量的统计分析above five pathways revealed that the expression of most in Z004 was downregulated, while the expression of most in Z011 was upregulated (Fig.4). Especially in the MAPK signalling pathway, which is closely related to the plant response to abiotic stress, the expression of all DEGs in Z004 was downregulated; however, in Z011, there were only 2 DEGs whose expression was downregulated, whereas that of the all other DEGs was upregulated (Fig.4d). In Z011, the number of DEGs was greatest after 24 h of NaCl treatment, and the number of DEGs was lowest after 72 h (Fig.4). The comparisons of Z011 and Z004 in different time points performed that the number of downregulated DEGs was decreased obviously after the salt treatment (Fig.4). In general, the number of DEGs involved in the salt-stress response in Z011 was significantly greater than that in Z004, and the number genes whose expression was upregulated obviously increased.

Identification of DEGs from comparisons of the Z004 and Z011 roots

度的缩小选择范围,我们focused on those within plant hormone signal transduction families, TF families and other gene families that have been reported to be involved in salt tolerance. A total of 233 DEGs were identified with a |log2(fold change)| ≥ 2 and padj < 0.05 in any one comparison and selected from six different comparisons of Z004 and Z011 roots. With respect to the hormone pathways, the ABA signalling pathway had the most DEGs (26 genes), followed by the auxin signal pathway (19 genes) (Fig.5a, Online Resource5). The DEGs in the ABA signalling pathway all belonged to theprotein phosphatase 2C(PP2C) family; however, in the auxin signalling pathway, 14 DEGs were related to auxin induction, and 5 DEGs were related to the auxin response (Fig.5a, Online Resource5). In addition, 6 DEGs in the ethylene signalling pathway belonged to theethylene insensitive 3(EIN3) family (Fig.5a, Online Resource5). With respect to TFs, a total of 8 TF families were identified. TheWRKYTF family had the most DEGs (37 genes), followed by thebHLHTF family (29 genes) (Fig.5b, Online Resource5). In addition, members of thebZIPTF family (22 genes),GRASTF family (18 genes),WD40TF family (12 genes),F-boxTF family (8 genes),TCPTF family (7 genes) andSBPTF family (5 genes) were identified (Fig.5b, Online Resource5). Moreover, a total of 25 genes in the DUF family, 13 ubiquitin genes and 6 heat-shock protein 70 (HSP70) gene were also identified (Fig.5c, Online Resource5).

From the above 233 DEGs, we selected 44 whose expression was significantly contrastingly up- or downregulated between the Z004 and Z011 roots after salt treatment (Table1). The expression of seven DEGs in the hormone signalling pathway was downregulated in the Z004 roots and upregulated in the Z011 roots. Among these DEGs, three belonged to the auxin-responsive proteinsmall auxin-up RNA(SAUR) family in the auxin signalling pathway, one belonged to the auxin response factor family in the auxin signaling pathway, one DEG belonged to theEIN3家庭在乙烯信号通路, and two DEGs belonged to thePP2Cfamily in the ABA signalling pathway. Six of these DEGs in the Z011 roots had the highest FPKM values at 24 h after salt treatment exceptnovel.25809, which suggested that these six genes might have significant effects on salt tolerance (Table1). In our research, members of various TF families involved in salt tolerance were identified. The major TF families identified were theWRKYandbHLHfamilies. TwelveWRKYand ninebHLHTFs are listed in Table1. The expression of all theWRKYwas downregulated in the Z004 roots and upregulated in the Z011 roots. Moreover, with the exception of that ofZjn_sc00039.1.g03650.1.am.mk(bHLH30), the expression of 8bHLHTFs was downregulated in the Z004 roots and 7 of them upregulated in the Z011 roots. In thebZIPTF family, six DEGs were selected, three of whose expression was upregulated in the Z004 roots and downregulated in the Z011 roots; andnovel.1352(bZIP53) had the opposite tendency of expression. In addition, the expression of threeGRASTF family members, twoWD40TF family members and oneSBPTF family member was downregulated in the Z004 roots and upregulated in the Z011 roots, exceptZjn_sc00016.1.g06200.1.am.mk(SCL9). The expression of theTCPTF family membernovel.30217(TCP7) was upregulated in the Z004 roots and downregulated in Z011 roots (Table1), and twoDUFfamily members,Zjn_sc00012.1.g05940.1.sm.mkhc(DUF315) andnovel.26532(DUF1671), were also selected and displayed a contrasting expression trend, which might indicate that they have opposite functions (Table1). From the above, the expression of 27 DEGs was downregulated in the Z004 roots and upregulated in the Z011 roots, and that of 4 DEGs was upregulated in the Z004 roots and downregulated in the Z011 roots, indicating that these genes might be related to the salt tolerance of zoysiagrass. Among them, the number of upregulated genes in the roots of the salt-tolerant accession Z011 roots was significantly greater than the number of downregulated genes.

Verification of the transcriptome sequencing data of the Z004 and Z011 roots

To verify the reliability of the transcriptome sequencing data of the Z004 and Z011 roots, twenty of the 39 DEGs whose expression was significantly contrastingly up- and downregulated signal pathway in the Z004 and Z011 roots after salt treatment were selected and validated via quantitative real-time PCR (qRT-PCR). With the exception ofZjn_sc00029.1.g04940.1.sm.mkhcandZjn_sc00011.1.g02540.1.sm.mk, approximately 90.0% of the DEGs were consistent between the RNA-seq and qRT-PCR data (Supplementary FigureS2). The results of the qRT-PCR and RNA-seq data were generally consistent, indicating that our transcriptome sequencing data of the Z004 and Z011 roots were reliable.

Phenotypic and physiological responses to salt stress inZ. japonicaSteud

Zoysiais a warm-season turfgrass that is widely used in home lawns, football fields and ecological management [50]。Zoysiagrass is recognized for its salt tolerance, hardiness, and drought tolerance and is suitable as a high-quality salt-tolerant turfgrass for landscaping in areas with saline soils [10]。Previous studies have shown that the salt tolerance ofZoysiais negatively correlated with the content of Na⁺and positively correlated with the content of K⁺in leaf fluids [25]。耐盐植物材料有很强的ability to maintain the K⁺/Na⁺ratio in their leaves and roots [25,33,34]。Our data from two accessions the (salt-sensitive material Z004 and salt-tolerant material Z011) with contrasting salt tolerances support these previous studies. After 350 mM NaCl treatment for 40 days, Z011 was more salt tolerant than was Z004, grew better, and had greater biomass (Fig.1a-e). Compared with those of Z004, the Na⁺concentrations in the leaves and secretions of Z011 were maintained at significantly lower levels, but there were no differences in the roots (Fig.2a-c). Moreover, there was no difference in K⁺concentration in the leaves of Z011 compared with Z004; however the K⁺concentration was significantly greater in the the roots of Z011, and the K⁺分泌也低的根源Z011(无花果.2d-f). These results indicated that, compared with Z004, Z011 may have improved salt tolerance by reducing the transport of Na⁺from the roots to the leaves, increasing the absorption of K⁺in the roots and reducing the secretion of K⁺from leaves to maintain a significantly greater K⁺/Na⁺ratio (Fig.2g, h).

Transcriptome sequencing of Z004 and Z011 and DEG identification in response to salt stress

Xie et al. [58] presented the first comprehensive transcriptome data ofZ. japonicaSteud. roots after 30 min of NaCl treatment, and a total of 32,849 unigenes and 4842 SSRs were identified. However, the important regulatory capabilities of the roots and leaves of this species under salt stress, and the key time point for salt tolerance regulation remain unknown. Our research involved the sampling of leaves and roots from Z004 and Z011 at 0 h, 1 h, 24 h and 72 h after treatment with 350 mM NaCl, and a total of 59,271 unigenes and 29,675 novel genes were revealed by RNA-seq. PCA revealed that the Z011 root samples had the highest dispersion degree, and the 24-h samples of the roots of both Z004 and Z011 were separated from those of other samples (Fig.3a). Moreover, the number of DEGs of Z011 roots was obviously more than that in the Z004 roots, and the number of DEGs at 24 h (Z01124R vs Z0110R) was 2.28 and 5.64 times that at 1 h (Z0111R vs Z0110R) and 72 h (Z01172R vs Z0110R) (Fig.3b, c, Supplementary FigureS1). Comparing the Z011 and Z004, the number of DEGs of roots was obviously more than leaves (Fig.3b). These results indicated that the roots might make a significant contribution to the difference in salt tolerance between Z004 and Z011 and the 24 h might be a relatively important time point for the salt-stress response.

Plant hormone signal transduction is involved in salt tolerance

Further evidence from the KEGG pathway enrichment analysis of the DEGs in the Z004 and Z011 roots demonstrated that plant hormone signal transduction has important effects on salt tolerance (Fig.4c). Seven DEGs involved in plant hormone signal transduction were selected, and the expression of six of them was downregulated in the Z004 roots and upregulated in the Z011 roots. Among these DEGs, oneSAUR41和两个SAUR77genes belonged to the auxin-responsive proteinSAURfamily in the auxin signalling pathway, oneARF6gene belonged to the auxin response factor (ARF) family in the auxin signaling pathway, oneethylene-insensitive-3-like-1(EIL1) gene belonged to theEIN3家庭在乙烯信号通路and onePP2C12and onePP2C27gene each belonged to thePP2Cfamily in the ABA signalling pathway (Table1).

SAURscompose a family of auxin-responsive genes that play an important role in the regulation of plant growth and development. However, the function of members of theSAURfamily in terms of salt tolerance has rarely been reported. Previous studies have shown only that the expression ofTaSAUR75is downregulated in wheat roots after salt stress, and increased root length, survival rate and expression of some salt and drought stress-responsive genes were detected inTaSAUR75-overexpressing transgenic plants compared with CK plants [13]。InArabidopsis, overexpression ofAtSAUR41promotes hypocotyl elongation and increases both primary root growth and lateral root number [19]。Yeast two-hybrid experiments showed thatAtSAUR77might participate in ethylene receptor signalling and promote plant growth [27]。However, the function of these two genes in salt tolerance has not yet been reported. In our research, under salt tolerance, the expression of bothZmSAUR41andZmSAUR77was downregulated in the Z004 roots and upregulated in the Z011 roots, indicating thatZmSAUR41andZmSAUR77might improve salt tolerance (Table1). ARF family bind to promoters of many auxin-regulated genes with auxin response elements (AuxREs, 5′ tgtctc 3′) and regulate the expression of auxin-induced genes [14,29]。InArabidopsis,ARF6andARF8are the target genes of miR167, and miR167 is induced by salinity and drought [20]。In our research, the expression ofZmARF6was downregulated in the Z011 roots after salt treatments, indicating thatZmARF6might negative regulated salt tolerance (Table1).

Ethylene considered a stress hormone involved in the plant response to salt tolerance.EIN3andEIL1are two ethylene-activated TFs and have been reported to be important for improving salt tolerance. InArabidopsis, accumulation of EIN3/EIL1 is induced by high salinity and likely enhances reactive oxygen species (ROS) scavenging at the seedling stage to promote salinity tolerance, as compared with wild-type plants,ein3 eil1double mutants have greater levels of ROS and lower levels of peroxidase-encoding transcripts and are hypersensitive to salt tolerance [38]。However, in contrast, overexpressionOsEIL1andOsEIL2in rice resulted in salt hypersensitivity at the seedling stage, and the lack ofOsEIL1andOsEIL2functionality increased salt tolerance. Furthermore, the negative regulation ofOsEIL1andOsEIL2in salt tolerance might contribute to the regulation ofhigh-affinity K⁺transporter 2;1expression and the absorption of Na⁺in roots [60]。In our research, under salt tolerance, the expression ofZmEIL1was downregulated in Z004 roots and upregulated in Z011 roots, indicating thatZmEIL1可能有一个积极的影响盐耐受性,which is consistent with the function ofAtEIL1inArabidopsis(Table1).

PP2Cenzymes are key elements involved in the ABA signalling pathway. In the present study, the expression levels ofPP2Cfamily genes differed in response to salt tolerance. OverexpressingAtPP2CG1(A. thaliana protein phosphatase 2C G Group 1) inArabidopsiscan enhance salt tolerance, whereas a lack ofAtPP2CG1function reduces salt tolerance. In addition,AtPP2CG1upregulated the expression levels of some stress-responsive genes under salt treatment, includingRD29A,RD29B,DREB2AandKIN1[31]。In maize, salt treatment induced intron methylation ofZmPP2Cand then significantly downregulated the expression ofZmPP2C[47]。Transcriptomic analysis ofCamellia sinensisrevealed that multiplePP2Cmembers participate in the salt tolerance response, such asPP2C2,PP2C3,PP2C14,PP2C51andPP2C60,whose expression was upregulated under salt treatment; however, the expression ofPP2C12,PP2C27andPP2C54was downregulated [52]。In our study, after salt treatment, the expression of bothZmPP2C12andZmPP2C27was downregulated in the Z004 roots and upregulated in the Z011 roots, indicating thatZmPP2C12andZmPP2C27might be positive regulators of salt tolerance (Table1).

TFs involved in salt tolerance

In our research, members of various TF families involved in salt tolerance were identified. Among them, the members of theWRKYandbHLHTF families were the most common (Table1). TheWRKYTF family is known to be involved in various physiological processes and many aspects of the plant defence system. We identified twelve differentially expressedWRKYgenes, and eight of them were reported to be related to salt tolerance. InGossypium raimondii, Cai et al. [3] reported thatWRKY35,WRKY40andWRKY64were induced by salt, and the expression levels were significantly upregulated. Moreover,AtWRKY40was found to be induced in response to salt stress inArabidopsis[42]。OverexpressingFcWRKY40ofFortunella crassifoliain tobacco and lemon increased salt tolerance, while silencingFcWRKY40decreased salt tolerance [8]。Wang et al. [55] cloned theWRKY56gene fromPopulus simonii×Populus nigra, which was significantly induced by salt treatment, and transformed it intoArabidopsis; the resultingWRKY56-overexpressing transgenicArabidopsisplants were more salt tolerant than were the wild-type plants, and the fresh weight and germination of the latter increased [55]。APsnWRKY70gene was also cloned fromP. simonii×P. nigraand confirmed the response to salt stress inPsnWRKY70-repressed plants, which exhibited enhanced salt tolerance [64]。In addition, AtWRKY70 has been reported to regulate salt stress by interacting with the Cys2/His2 zinc finger protein Zat7 [7]。WRKY71andWRKY28are homologues and are induced by high salinity. Overexpression ofWRKY71orWRKY28resulted in insensitive flowering ofArabidopsisplants in response to high salinity, while downregulated ofWRKY71andWRKY28resulted in more sensitive flowering ofArabidopsis[62]。Given that the expressions ofWRKYsin our research were mainly downregulated in the Z004 roots and upregulated in Z011 roots,WRKYgenes may play positive roles in salt tolerance.

ThebHLHTF family has been reported to participate in the regulation of abiotic stress-related signal transduction. Nine differentially expressedbHLHgenes were identified in our research, and five of them have been reported to be regulated by NaCl (Table1). ThebHLH30genes in chrysanthemum, evergreen tree and upland cotton are induced under salt stress and have been reported to increase salt tolerance in yeast [6,45,56]。InArabidopsis, two presumptive paralogues ofbHLH92,bHLH41andbHLH42,are induced by salt treatment [16]。Moreover, transcriptome analysis of the salinity tolerance ofBrassica juncearevealed that the expression ofbHLH101was downregulated under salt treatment [44]。In our research, the expressions of thebHLH13,bHLH41,bHLH92,bHLH94,bHLH96andbHLH101genes were downregulated in the Z004 roots and upregulated or maintained in the Z011 roots, indicating that thesebHLHgenes might be positive regulators of salt tolerance. Among them, the expression trend ofbHLH101inZoysiawas opposite that inB. juncea[44], indicating thatbHLH101may have different functions in different species. In addition,bHLH30, whose expression was upregulated in the Z004 roots and downregulated in the Z011 roots, might play a negative role in the salt tolerance ofZoysia,which contrasts with the results of previous reports.

Members of thebZIPTF family have important roles in many biological processes, and somebZIPTF family members have been reported to exert biological functions under salt stress. Transgenic plants overexpressingAtbZIP1,ZmbZIP72,GmbZIP132andZmABP9presented enhanced tolerance to salt stress [28,46,53,61]。Six differentially expressedbZIPgenes were identified in our research, andbZIP2,bZIP44andbZIP53were reported to be involved in salt tolerance (Table1). The transcript level of theLebZIP2gene inLycopersicon esculentumincreased after salt-stress treatments, and thebZIP1andbZIP53mutants displayed reduced salt tolerance [15,43]。OverexpressingGmbZIP44gene could increase the salt tolerance of transgenic plants in soybean [28]。In our research, the expression ofTGAL6,bZIP2,bZIP11andbZIP44was upregulated in the Z004 roots and downregulated in the Z011 roots, while the expression ofbZIP53was downregulated in the Z004 roots and upregulated in the Z011 roots, indicating that different bZIP members might play different roles in the salt tolerance process inZ. japonica.

Among the remaining seven TFs (Table1), only the salt tolerance function of theRACK1Agene has been clearly elucidated. In rice, suppression ofOsRACK1Aincreased salt tolerance by maintaining low Na⁺and high K⁺concentrations in both the roots and leaves [63]。Our data showed that the expression of threeGRASTF family members (SCL9andNSP1), twoWD40TF family members (WDR44andRACK1A) and oneSBPTF family member (SPL15) was downregulated in the Z004 roots and upregulated in the Z011 roots, while the expression of oneGRASTF family member (SCL9) and oneTCPTF family member (TCP7) was upregulated in the Z004 roots and downregulated in the Z011 roots. Thus, these genes may have important roles in salt tolerance, but their functions require additional research.

TheDUFfamily is involved in the salt tolerance ofZ. japonica

DUF family members compose a large number of uncharacterized protein families within the Pfam database (http://pfam.xfam.org/family), which contains approximately 3000 families [1]。Some DUF proteins are active in plant development [2,4], and other members ofDUFfamilies are involved in the stress response [18,54], especially the salt-stress response. TheAhDGR2gene inAmaranthus hypochondriacusencodes a DUF642 domain-containing protein, and plants overexpressingAhDGR2present increased sensitivity to NaCl treatment [37]。OsDSR2, which encodes a DUF966 domain-containing protein, also negatively regulates salt stress in rice [32]。However, overexpressingSIDP361(a DUF1644 protein-coding gene) in rice significantly enhances salt tolerance at both the seedling and heading stages [24]。The expression ofOsDUF810.7significantly increases under salt treatment, and overexpression of this gene inE. coliimproves the salt tolerance of the bacterium [23]。TwoDUFfamily members,ROPGEF7(a DUF 315 protein-coding gene) andUFSP(a DUF 1671 protein-coding gene), were selected in our research and showed an inverse expression trend (Table1), indicating that these two DUF genes might play important roles in the salt tolerance process inZ. japonicaand might have contrasting functions.

Our research performed that salt-tolerantZ. japonicaaccession Z011 may have improved salt tolerance by reducing Na⁺transport from the roots to the leaves, increasing K⁺absorption in the roots and reducing K⁺secretion from the leaves to maintain a significantly greater K⁺/Na⁺ratio. Twenty-four hours might be a relatively important time point for the salt-stress response of zoysiagrass. The auxin signal transduction family, ABA signal transduction family,WRKYTF family andbHLHTF family may be the most important families inZoysiasalt-stress regulation. This study provides fundamental information concerning the salt-stress response ofZoysiaand improves the understanding of molecular mechanisms in salt-tolerant plants.

Plant materials and treatment

On the basis of the salt tolerance of 206 zoysiagrass accessions identified by their leaf firing, relative shoot clipping dry weight, verdure dry weight and root dry weight (H. L. Guo, unpublished data), the salt-sensitiveZ. japonicaaccession Z004 and the salt-tolerantZ. japonicaaccession Z011 were used in this study. Both accessions were collected in 1995 by Jianxiu Liu, turfgrass major of Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, China. Z004 was collected in a wild grassland in Lushan area (Jiangxi, China, 28°36′N, 116°00′E) and Z011 was collected in a hilly land of Lanxi area (Zhejiang, China, 29°13′N, 119°30′E). AsZoysiaWilld。is not endangered, collection of samples for scientific purposes was permitted by local legislation. Professor Shouliang Chen, taxonomy major, and Jianxiu Liu, turfgrass major of Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, undertook the formal identification of the samples according toflora of Reipublicae Popularis Sinicae(Vol.10, No.1, 1990). Previous morphological and DNA analyses also confirmed the correct identification of the two accessions [12,30]。Both accessions were maintained at an experimental field of the Institute of Botany, Jiangsu Province and Chinese Academy of Sciences. Twenty uniform sprigs of both materials were transplanted from the experimental field of the Institute of Botany were planted into 9-cm-diameter and 6-cm-deep plastic pots filled with coarse silica sand. Ten pots were planted per treatment and material. The pots were suspended over tanks (66.56 × 45.56 × 17.0 cm³) filled with 45 L of 1/2-strength Hoagland’s solution. The grasses were clipped weekly until growth was consistent and were cultivated in a greenhouse with a day/night temperature of 35/28 °C, 16 h of light/8 h of dark, 75% relative humidity and 800 μmol m^− 2 s^− 1of photosynthetically active radiation. NaCl treatment (350 mM) was initiated after 2 months of cultivation.

Observations and measurements of the salt tolerance ofZ. japonicaSteud

After the turfgrasses were subjected to salt treatment for 40 days, the leaf firing was assessed by visual rating via a scale of 1 (slight firing) to 9 (severe firing). All the treated grasses and CK grasses were divided into three parts: shoot clippings, verdure and roots. Among them, the part of the grass blades clipped to a height of 4.0 cm were considered shoot clippings, and the part excluding the shoot clippings and roots was considered the verdure. These three parts were dried at 70 °C for 48 h, after which the dry weight was determined. The relative dry weights were then calculated according to the following equation: relative dry weight = Wt/Wo × 100%, where Wt is the dry weight in the NaCl group, and Wo is the dry weight in the CK group. Three biological replicates were tested, and data from individual measurements were averaged and analysed via SPSS statistical software 18.0 (ttest) (SPSS Inc., Chicago, IL, USA).

Determination of the concentrations of Na⁺and K⁺

To determine the concentrations of Na⁺and K⁺in the leaves and roots, 20 mg of dry powder samples was placed into sealed test tubes that contained 15 ml of ddH₂O. All the samples were boiled for 1 h and incubated at room temperature for 24 h. After the samples were filtered, the extracted Na⁺and K⁺concentrations were measured by flame photometry (Model FP6410; Shanghai Xinyi Instruments Inc., Shanghai, China) and calculated on the basis of the tissue dry weight (mmol kg^− 1). To determine the secretion of Na⁺and K⁺, the leaves were washed with ddH₂O 3 times after 5 days of salt treatment to remove the salt crystals secreted from the leaf surface. Seven days later, 4–8 pieces of mature leaves were removed and placed into centrifuge tubes. Each centrifuge tube was supplemented with 10 ml of ddH₂O and shaken for 5 s to fully dissolve the salt crystals on the leaf surface into the ddH₂O. Afterward, the leaves in the centrifuge tubes were removed and dried at 70 °C for 48 h, after which the dry weight was determined. The Na⁺and K⁺concentrations within 10 ml of ddH₂O were measured by flame photometry and calculated on the basis of the corresponding leaf dry weight (mmol kg^− 1). The K⁺/Na⁺ratio was considered the ratio of the K⁺concentration to the Na⁺concentration. Three biological replicates were analysed, and the data from the individual measurements were averaged and analysed using SPSS statistical software 18.0 (ttest) (SPSS Inc., Chicago, IL, USA).

RNA-seq

The leaf and root tissues of Z004 and Z011 at four time points (0 h, 1 h, 24 h, and 72 h) after salt treatment were sampled, frozen in liquid nitrogen and stored at − 80 °C. Each collected sample was 0.1 g, and three biological replicates were analysed. The total RNA was extracted [58),每三个生物复制RNA样本s were mixed fully into one RNA library. A total of 16 RNA libraries were generated after the samples were pooled: Z0040L (Z004, 0 h, leaf), Z0041L (Z004, 1 h, leaf), Z00424L (Z004, 24 h, leaf), Z00472L (Z004, 72 h, leaf), Z0040R (Z004, 0 h, root), Z0041R (Z004, 1 h, root), Z00424R (Z004, 24 h, root), Z00472R (Z004, 72 h, root), Z0110L (Z011, 0 h, leaf), Z0111L (Z011, 1 h, leaf), Z01124L (Z011, 24 h, leaf), Z01172L (Z011, 72 h, leaf), Z0110R (Z011, 0 h, root), Z0111R (Z011, 1 h, root), Z01124R (Z011, 24 h, root), and Z01172R (Z011, 72 h, root). The cDNA library construction and transcriptome sequencing were performed by Novogene (Tianjin, China) (http://www.novogene.com/) on an Illumina HiSeq™ 2000 platform, and 125 bp/150 bp paired-end reads were generated. The datasets are available in the NCBI repositoryhttp://www.ncbi.nlm.nih.gov/bioproject/PRJNA559944.

De novo assembly, gene expression levels and identification of DEGs

Clean reads were obtained from the raw data by the removal of reads containing adapters, reads with ambiguous ‘N’ bases and reads of low quality. The Q20, Q30 and GC content of the clean reads were calculated, and the subsequent analyses were based on the high-quality clean reads. De novo assembly of theZ. japonicatranscriptome from the reference genome of zoysiagrass [48] was accomplished via Hisat2 (v2.0.5) [17]。The mapped reads of each sample were then assembled, and the novel transcripts were predicted by StringTie (v1.3.3b) [39]。The gene expression levels were calculated according to the FPKM method [36]。The edgeR R package (3.18.1) [41] was subsequently used to analyse significant differences in expression with padj < 0.05 and fold change > 2, and DEGs were identified with a |log2(fold change)| > 1 and padj < 0.05.

GO and KEGG enrichment analyses of DEGs

All the DEGs were mapped to terms identified from the GO and KEGG (http://www.genome.jp/kegg/) enrichment analyses, and the clusterProfiler R package was used to analyse the GO enrichment of the DEGs and to test the statistical enrichment of the DEGs in the KEGG pathways. The GO terms and KEGG categories with padj < 0.05 (correctedP-value) were selected as significant GO classifications and KEGG pathways for the DEGs in each comparison.

qRT-PCR validation of transcription

Twenty DEGs were selected from the 44 DEGs whose expression was significantly contrastingly up- or downregulated in the Z004 and Z011 roots after salt treatment to validate the reliability of the transcriptome data. The primers of the DEGs were designed using Primer 5.0 software, andZjActin(GenBank: GU290545.1) was used as a housekeeping gene. Each sample consisted of three biological replicates, and the qRT-PCR assays were carried out as described by Xie et al. [58]。The gene primers used are listed in Online Resource6.

The sequencing data are available in the NCBI repositoryhttp://www.ncbi.nlm.nih.gov/bioproject/PRJNA559944. The datasets analyzed during the current study are available from the corresponding author on reasonable request. All data generated or analyzed during this study are included in this published article [and its Additional files].

cDNA:

Complementary DNA

CK:

Control group

DEGs:

Differentially expressed genes

FPKM:

Fragments per kb per million fragments

GO:

Gene ontology

KEGG:

Kyoto Encyclopedia of Genes and Genomes

NCBI:

National Center for Biotechnology Information

PCA:

Principal component analysis

qRT-PCR:

Quantitative real-time PCR

RNA-seq:

RNA sequence

TF:

Transcription factors

Tr:

The treatment group

We thank Novogene Company (Beijing) for helping with transcriptome sequencing.

This work was funded by the National Natural Science Foundation of China [31572155, 31672195], the Natural Science Foundation of Jiangsu Province, China [BK20151371], and National Key R&D Program of China [SQ2016FY326000402].

Affiliations

1. 江苏省植物研究所Chinese Academy of Sciences, Nanjing, 210014, China

   Jingjing Wang, Cong An, Hailin Guo, Xiangyang Yang, Jingbo Chen, Junqin Zong, Jianjian Li & Jianxiu Liu

Contributions

HG, XY, JC and JZ performed the physiological experiments. JW, CA and JJL performed the transcriptomic analyses and verification experiments. HG and JXL designed the experiment. JW and HG participated in writing the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence toHailin Guo.

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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