Abstract Background Areas with saline soils are sparsely populated and have fragile ecosystems, which severely restricts the sustainable development of local economies. Zoysia grasses 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 of Zoysia species remains unknown. Results The phenotypic and physiological responses of two contrasting materials, Zoysia japonica Steud. 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 the DUF family. Conclusions Zoysia salt 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, WRKY TF family and bHLH TF family may be the most important families in Zoysia salt-stress regulation. Keywords: DUF, Hormone signal, Salt tolerance, Transcription factor, Transcriptome, Zoysia grass Background Soil salinization is a worldwide problem. Areas 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. Zoysia Willd. is a genus of perennial plants belonging to the family Poaceae, subfamily Chloridoideae, tribe Zoysieae [[39]51]. Zoysia grasses 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 [[40]10]. Compared with other Poaceae family members, Zoysia grasses have received less attention in the research community. However, as an alternative grass species for landscaping in saline-alkali soil, Zoysia has superior growth qualities [[41]26]. In particular, among the three most important commercial species, Zoysia japonica Steud. is distinctly tolerant to abiotic stress [[42]51]. Therefore, studying the salt tolerance of Zoysia plants is highly important. Previous studies on salt tolerance of Zoysia mainly focused on the evaluation of salt tolerance and the physiological mechanisms governing salt tolerance. Salt tolerance evaluations have shown that the salt tolerance of Zoysia plants has rich genetic variation [[43]25, [44]35, [45]40, [46]57]. This variation makes for convenient selection of materials with contrasting salt tolerances for studying the salt tolerance mechanism of Zoysia. Zoysia plants secrete salt; all Zoysia plant leaves have salt glands that regulate ion balance by selectively secreting salt ions. The salt tolerance of Zoysia plants is positively correlated with the rate of Na^+ secretion from salt glands in leaves and the density of salt glands per unit leaf area [[47]21, [48]22, [49]33]. Moreover, previous studies have shown that the salt tolerance of Zoysia is 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 of Zoysia [[50]25, [51]33, [52]34]. The salt tolerance of Zoysia is a very important trait, but to date, its molecular regulatory mechanism remains unknown. The Na^+/H^+ antiporter gene ZjNHX1, which belongs to the plant NHX-gene family, was cloned from Z. japonica, and studies have shown that ZjNHX1 plays an important role in ion homeostasis and salt tolerance [[53]9]. In addition, the glycine-rich RNA-binding protein-coding gene ZjGRP was isolated from Z. japonica and was strongly induced by NaCl treatment. ZjGRP-overexpressing Arabidopsis thaliana plants present low germination rates, slow seedling growth and poor salt tolerance [[54]50]. ZjZFN1 is a C[2]H[2]-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. Overexpressing ZjZFN1 in A. thaliana can improve seed germination and increase salt tolerance by improving the transcriptional activities of several salt-tolerance-related genes under salt stress [[55]49]. Studies on the salt tolerance genes of Zoysia are scarce. However, using a full-length cDNA expression library in yeast, Chen et al. [[56]5] systematically excavated the salt tolerance genes in Zoysia matrella and 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. [[57]58] presented the first comprehensive transcriptome data of Z. japonica Steud. 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 the AP2/EREBP family, bZIP family, NAC family, WRKY family, MYB family and bHLH family play significant roles in the early response to salt stress [[58]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 [[59]11, [60]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. japonica Z004 (salt sensitive) and Z011 (salt tolerant), in response to salt stress. On the basis of the existing Zoysia reference genome [[61]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 the DUF family. Thus, our research provides fundamental information for use in future salt-stress studies of Zoysia and improves the understanding of molecular mechanisms in salt-tolerant plants. Results Phenotypic and physiological responses of Z. japonica Steud. To salt stress Japanese lawngrass (Z. japonica Steud.) 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 of Z. 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. [62]1a). Moreover, the leaf firing of Z004 was significantly greater than that of Z011 (Fig. [63]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. [64]1c-e). Fig. 1. [65]Fig. 1 [66]Open in a new tab Phenotypic response of Z. japonica Steud. to salt stress. a Two materials with contrasting salt tolerances, Z004 (salt sensitive) and Z011 (salt tolerant), were exposed to 350 mM NaCl for 40 days. b The leaf firing of the Z004 and Z011 grasses after NaCl treatment for 40 days. c The relative shoot clipping dry weights of Z004 and Z011 after NaCl treatment for 40 days. d The relative verdure dry weights of Z004 and Z011 after NaCl treatment for 40 days. e The relative root dry weights of Z004 and Z011 after NaCl treatment for 40 days. The values are presented as the means ± SEs. The asterisks above the bars indicate significant differences between the respective values (p < 0.05) 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. [67]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. [68]2a-c). In the roots of Z004 and Z011 after NaCl treatment, the Na^+ concentrations were not different (Fig. [69]2b). However, in the leaves, the Na^+ concentrations and secretions were significantly lower in Z011 than in Z004 (Fig. [70]2a, c). Fig. 2. [71]Fig. 2 [72]Open in a new tab Physiological response of Z. japonica Steud. to salt stress. a The Na^+ concentration in Z004 and Z011 leaves after Ck and NaCl treatment. b The Na^+ concentration in Z004 and Z011 roots after Ck and NaCl treatment. c Na^+ secretion by Z004 and Z011 after Ck and NaCl treatment. d The K^+ concentration in Z004 and Z011 leaves after Ck and NaCl treatment. e The K^+ concentration in Z004 and Z011 roots after Ck and NaCl treatment. f K^+ secretion by Z004 and Z011 after Ck and NaCl treatment. g The K^+/Na^+ ratio in Z004 and Z011 leaves after Ck and NaCl treatment. h The K^+/Na^+ ratio in Z004 and Z011 roots after Ck and NaCl treatment. The values are presented as the means ± SEs. The asterisks above the bars indicate significant differences between the respective values (p < 0.05) 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. [73]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. [74]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. [75]2f). Comparing with Z004, Z011 maintained a greater K^+/Na^+ ratio in both the leaves and roots (Fig. [76]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 Resource [77]1). First of all, the total reads of our RNA-seq were mapped to the rice and sorghum genomes as references via Hisat2