MsaH2A.W is identified response to salt tolerance in Miscanthus sacchariflorus

Miscanthus is a perennial forage plant with great potential for high stress tolerance and biomass yield. It has strong adaptability for growing in saline land and avoids competition with grain crops in arable lands. However, little is known about the underlying genetic basis of Miscanthus adaptation to salt stress. Two diploid species of the genus Miscanthus, Miscanthus sinensis and Miscanthus sacchariflorus, were the focus of this study. The transcriptome variations of these varieties and their hybrid were analysed using RNA‐seq technology under salt treatment. The number of differentially expressed genes in M. sinensis was much higher than that in M. sacchariflorus and their hybrid under salt stress, which indicated that M. sacchariflorus and their hybrid require less transcriptional variation. In addition, most salt‐tolerant genes in the enriched salt‐tolerant pathways were induced in the roots of M. sinensis and constitutively highly expressed in the roots of M. sacchariflorus and their hybrid under salt stress. According to this expression pattern of known salt‐tolerant genes, a histone variant gene MsaH2A.W of M. sacchariflorus was mined and consequently proved for the first time that it could enhance the salt tolerance of transgenic Arabidopsis plants. Overall, this study provides valuable genetic resources for studying the underlying genetic basis of salt stress resistance in Miscanthus. Identification of the salt tolerance gene MsaH2A.W can promote the genetic improvement and molecular breeding of salt‐resistant species.


| INTRODUCTION
Salt stress is a major abiotic stress factor that threatens plant agricultural production worldwide (Hazell & Wood, 2008). Approximately 45 million hectares of the world's arable land are affected by salinity (Shrivastava & Kumar, 2015). High intensity and long periods of salt stress change the physiological processes and morphological structure of plants, which stunts the growth and development of plants and may even lead to death (Paul, 2013). To maintain basic life activities under salt stress, plants alter their specific cellular and molecular activities and physiological and biochemical processes by activating a large number of salt tolerance genes and accumulating the corresponding salt tolerance proteins (Van et al., 2020;Zhao et al., 2021). Therefore, it is of great importance to comprehensively study the salt tolerance mechanism for the genetic improvement of salt resistance of plants.
To resist salt stress in the external environment, plants can activate diverse physiological, biochemical and molecular tolerance mechanisms to maintain basic physiological activity (Gong, 2021;Zhu, 2002). Antioxidant enzymes and non-enzymatic compounds play a key role in ROS detoxification under salt stress (Bohnert & Sheveleva, 1998;Gupta & Huang, 2014). The improvement of antioxidant enzyme activity can effectively enhance the salt tolerance of plants (Roxas et al., 2000;Xu et al., 2015;Yang et al., 2021). Based on the osmotic signalling pathway, plants can efficiently alleviate osmotic stress by regulating specific gene expression, accumulating osmolytes (proline, sugar and polyol) and activating water transport systems (Shumilina et al., 2019). The interaction of several phytohormones, such as abscisic acid (ABA), auxins (indole acetic acid), jasmonic acid (JA), cytokinins, salicylic acid and brassinosteroids, could participate in the salt stress response and regulate the expression of salt tolerance-related genes by transmitting stress signals (Ma et al., 2006;Osthoff et al., 2019). In addition, stress-activated transcription factors (TFs), such as WRKY and NAC (NAM, ATAF1/2 and CUC2), play a crucial role in the regulatory pathway of stress-related gene expression together with other abiotic stress response factors (Gao et al., 2020;Li et al., 2021;Shinozaki & Yamaguchi-Shinozaki, 1997). At present, some research on ROS, plant hormone signalling pathways and TFs has advanced the study of the molecular mechanism of multiple species in response to salt stress. However, research on the salt stress tolerance of Miscanthus crops is very limited.
Miscanthus, as a major perennial C4 grass, belongs to the Saccharinae subtribe of the Andropogoneae tribe (Poaceae) (Paterson, 2012). In this subtribe, Miscanthus, Saccharum and Sorghum have been extensively researched for their beneficial characteristics of bioenergy production. Compared with sorghum and sugarcane, Miscanthus possesses the most cellulose/hemicellulose per unit area of bioenergy production (Zhang, Ge, et al., 2021). There are great differences in lignocellulose components in stems of 179 species of Miscanthus, which indicates that the lignocellulose components of Miscanthus resources in China are rich in genetic diversity . In addition, Miscanthus has good paper-making properties (Miao et al., 2021), can be used as high-quality feed and raw material for edible fungi production (Finet et al., 2021), and has important economic value and ecological significance as an ornamental plant (Mitros et al., 2020). The cultivated land area is increasing to meet the increasing demand for grain (Heaton et al., 2008). Hence, Miscanthus has been widely researched and applied (Ge et al., 2019;Li et al., 2014;Sun et al., 2014). The genomes of multiple genera of Miscanthus have been well described, such as Miscanthus floridulus (Zhang, Ge, et al., 2021), Miscanthus lutarioriparius (Miao et al., 2021) and Miscanthus sinensis (Mitros et al., 2020). These genome data are helpful for genetic improvement and functional genomic research of Miscanthus. To date, most research on Miscanthus has mainly focused on the utilization of biomass, cellulose/hemicellulose and forage (Bukowski et al., 2020;Dai et al., 2022;Golfier et al., 2017;Lee & Kuan, 2015;Xu et al., 2020). In addition, Miscanthus, as a perennial wild grass, has strong adaptability and potential to grow in marginal land such as saline land to avoid competing for arable lands with grain crops (Fu et al., 2009;Wang, Shao, et al., 2011). Studies have shown that Miscanthus can maintain a relatively high growth rate and biomass accumulation compared to other plants under salt stress (Plazek et al., 2014;Stavridou et al., 2017). Miscanthus as a moderately salt-tolerant crop, acquired a wide range of stress-responsive genes during long-term adaptation to marginal lands (Hung et al., 2009;Ogura & Yura, 2008). Chen et al. (2017) showed that 70 Miscanthus genotypes had broad diversity of salt tolerance, and several highly salt-tolerant genotypes with different mechanisms could be used as valuable breeding materials (Chen et al., 2017). By comparative transcriptomic analysis of five Miscanthus populations under long-term salt stress, 59 genes shared in five populations were found to respond to high-salinity environments, and approximately 70% of them have been proven to be associated with abiotic stress responses (Song et al., 2017). Studies have shown that the NAC TFs of Miscanthus, such as MINAC9, MINAC10 and MINAC12, could improve the salt tolerance of transgenic Arabidopsis plants (He et al., 2019;Yang et al., 2018;Zhao et al., 2016). At present, only a few studies have been performed on the salt tolerance of Miscanthus, and the underlying genetic basis of salt stress tolerance in Miscanthus germplasm has not been deeply studied.
In eukaryotes, histones are the basic components of chromatin structure. Several kinds of histones, including H2A, H2B, H3 and H4, interact to constitute the nucleosome octamer core (Kornberg, 1974;Sun et al., 2014). Histone variants play a key role in genome integrity, chromosome segregation and gene expression (Yelagandula et al., 2014). In plants, altered activity or levels of histone variants are associated with abiotic stress responses (Nguyen & Cheong, 2018;Talbert & Henikoff, 2010). The linker histone variant HIS1-3 of Arabidopsis is induced by high-salinity and drought stress (Ascenzi & Gantt, 1999;Wu et al., 2022). Under drought stress, H1-S transgenic tomato plants were obtained by an antisense strategy, and H1-S could promote stomatal closure and consequently enhance drought resistance (Scippa et al., 2004). Similarly, the histone variant TaH2A.7 of wheat could also improve drought resistance by promoting stomatal closure (Xu et al., 2016). The histone variant H2A.Z participates in environmental temperature perception and nutrient stress response in Arabidopsis (Kumar & Wigge, 2010;Smith et al., 2010). The H3.2 variant gene RH3.2A of rice is involved in the response to salt stress (Qiu et al., 2006). Although several histone variants such as H1-S and H2A.Z, H2A.7 and H3.2A have been proven to respond to abiotic stress, whether there are more histone variants involved in abiotic stress response has not been elucidated (Chang et al., 2020;Zhu et al., 2013).
Here, the transcriptomes of M. sinensis, M. sacchariflorus and their hybrid under salt treatment were compared and analysed. The salt resistance strategies and expression patterns of salt tolerance genes in these three species under salt stress were investigated, and their different underlying genetic basis of salt tolerance was explored. Then, based on the expression pattern of salt-tolerant genes, we identified a histone variant gene MsaH2A.W, and first proved that its overexpression enhanced the salt tolerance of transgenic Arabidopsis plants. This study provides researchers with valuable genetic resources related to salt tolerance, and lays a genetic basis for the development and cultivation of new salt-tolerant varieties.

| Plant materials, growth conditions and stress treatment
Miscanthus sinensis (M009) and Miscanthus sacchariflorus (M350) were collected from Hunan and Gansu, China, respectively. Both were diploid (2n = 2× = 38). M009 and M350 were transplanted into the Miscanthus germplasm resource nursery of the Agricultural Experiment Station of Shandong Agricultural University, and the hybrid (M374) was obtained by crossing M009 and M350. M374 also grew in the same location. The rhizomes of M009, M350 and M374 were separated into single buds, and each single bud was transferred to a plastic pot with perlite and grown in a greenhouse with 1/2 Hoagland solution, a 16-h light/8-h dark cycle at a mean temperature of 26°C ± 1°C and a relative humidity of 50%. When the seedlings grew to the three-leaf-age, the salt-treatment groups were cultured with 1/2 Hoagland solution (200 mM NaCl), and the control groups were irrigated in 1/2 Hoagland solution as before. After 12 h of treatment, the roots and leaves of the salt-treatment groups and control groups were collected for RNA-seq. Two biological replicates were set up from the salt-treatment groups and control groups of M009, M350 and M374. The phenotypes of the three species were identified after 28 days of salt stress treatment.

| RNA isolation, cDNA library preparation and sequencing
TRIzol reagent (Invitrogen) was used for RNA isolation of all samples. DNaseI was used to eliminate DNA contamination in RNA samples. The concentration and quality of each RNA sample were measured by Nanodrop 2000c spectrophotometers and 0.8% agarose gel electrophoresis. The custom high-throughput Illumina library method was followed to prepare RNA-seq libraries. The quality of RNAseq libraries was measured by Qubit 2.0 Fluorometer (Life Technologies), Agilent Bioanalyzer 2100 system and 3% agarose gel electrophoresis. The 2 × 150 bp paired-end configuration was used for sequencing the cDNA libraries, and the lengths of the fragments were approximately 300-500 bp. The HiSeq 4000 system of Illumina was adopted for carrying out sequencing.

| De novo assembly of transcripts
Trimmomatic was used for initially filtering raw reads by removing adapter-containing reads, low-quality reads and empty reads (Bolger et al., 2014). Then, the clean reads with two biological repeats of roots and leaves were combined from the control and salt treatment groups. Trinity was used to assemble the de novo transcriptome (Grabherr et al., 2011). RSEM was used to accurately quantify the transcripts in the combined data (Li & Dewey, 2011) and calculate transcripts per kilobase million (TPM) to represent the transcript abundance of each unigene. Sequences with TPM values less than one were filtered out from the raw assembled transcripts. In addition, cd-hit software was used to remove redundant sequences with similarity greater than 90%. Finally, three transcriptomes corresponding to M009, M350 and M374 were used as reference transcriptomes separately.

| Analysis of differentially expressed genes
The TPM of each gene was obtained by mapping the clean data of each sample to the reference transcriptome. By comparing the expression levels of each gene between the salt treatment groups and control groups, the differentially expressed genes (DEGs) of M009, M350 and M374 were screened based on the differential significance criteria (|log2FC | ≥ 1 and FDR <0.01).

| GO functional annotation and KEGG enrichment analysis
To obtain the gene annotation of each transcript, unigenes were aligned with the UniProt database (http://www.unipr ot.org/) by means of local BLAST. The GO annotations and KO annotations were acquired by loading the alignment results into the Trinotate sqlite database. The clusterProfiler package of R was used to identify GO terms that annotated a series of enriched genes with a p < 0.01 and the KEGG pathway that was enriched by DEGs. The metabolic pathways with p < 0.05 were screened in three species under salt stress.

| The mining strategy of salt-tolerant genes
The amino acid sequences of the three reference transcriptomes were aligned to obtain the orthologous transcripts of M009, M350 and M374 by proteinortho6. pl software. By analysing the expression patterns of orthologous DEGs of the three species under salt treatment, the adaptation strategy of Miscanthus to a high salt environment could be elucidated. Then, based on the expression patterns of known salt tolerance genes, candidate salt tolerance genes could be identified.

| Validation of RNA-seq data by qRT-PCR
To verify the reliability of the transcriptome data, six known salt tolerance genes including P5CS, GST, NIP, TIP, JIP and CaM, and one excavated salt tolerance gene, H2A.W were chosen for qRT-PCR analysis. Total root RNA of M009, M350 and M374 was isolated using TRIzol reagent. cDNA was synthesized using an Evo M-MLVPlus cDNA Synthesis Kit (Accurate Biology). According to the manufacturer's instructions, qRT-PCR was performed with the Bio-Rad CFX96 Real-Time instrument by using UltraSYBR Mixture (Low ROX) reagent. The expression levels of seven genes were normalized to those of the internal control. Three biological replicates were performed for each gene. Gene primers are listed in Table S2.

Arabidopsis
To verify that MsaH2A.W could enhance the salt tolerance of plants, the MsaH2A.W coding sequence was cloned into the pCAMBIA3300-3flag vector from M. sacchariflorus cDNA to generate 35S::MsaH2A.W-3flag constructs. Agrobacterium tumefaciens GV3101 with the 35S::MsaH2A.W-3flag vector was used to infect Arabidopsis plants by floral dip. Overexpression of MsaH2A.W was performed in wild-type (WT) Arabidopsis (ecotype Col-0). Transgenic Arabidopsis plants were screened on 1/2 MS medium with 0.005% Basta. Two independent transgenic Arabidopsis lines (OE2 and OE7) were confirmed according to semiquantitative RT-PCR and consequently selected for phenotypic analysis. Primers are shown in Table S3.

Arabidopsis plants
To investigate the salt tolerance of two transgenic Arabidopsis plants, sterile seeds of WT, OE2 and OE7 were sown on 1/4 plant nutrient solution (PNS; Gong et al., 2004) solid medium. After 5 days of vernalization at 4°C, these seeds were vertically cultivated at 22°C with a 16-h light/8-h dark cycle. Five-day-old seedlings were transferred to 1/4 PNS solid medium with or without 100 mM NaCl for salt sensitivity analysis. Phenotypic analysis was performed after 10 days. Furthermore, the entire primary root length of seedlings was measured by ImageJ software, and statistical significance was analysed. There were three biological replicates of the WT and transgenic Arabidopsis lines in the salt treatment experiments.

M374 under salt stress
To study the resistance of M009, M350 and M374 to salt stress, their well-cultured seedlings were treated with 200 mM NaCl for 28 days. The leaf wilting degree of M009 was higher than that of M350 and M374, and M350 and M374 clearly grew better than M009 after salt treatment. Moreover, substantial chlorosis of leaves was observed in M009 ( Figure S1). These observations showed that M350 and M374 had stronger salt tolerance than M009.

| De novo assembly and evaluation of transcriptomes
To investigate the underlying mechanism of different salt tolerances in M009, M350 and M374, RNA-seq was performed through next-generation sequencing (NGS). Three reference transcriptomes corresponding to M009, M350 and M374 were acquired based on de novo transcriptome assembly. A total of 71,719, 76,882 and 61,879 unigenes were assembled in M009, M350 and M374, respectively. The N50 of unigenes was 1469, 1406 and 1459 bp, and their GC content was 52.48%, 52.10% and 52.56% in M009, M350 and M374, respectively (Table 1). The clean reads of each sample were mapped to their corresponding unigenes, and the average mapping ratios of M009, M350 and M374 were 91.10%, 90.57% and 89.37%, respectively. Then, the distribution of unigene length in the M009, M350 and M374 assembly results was analysed. The sequence lengths of 46.03%, 42.32% and 49.13% unigenes from M009, M350 and M374 were over 600 bp ( Figure S2). These results suggested that the transcriptomes of M009, M350 and M374 were eligible to be used as reference transcriptomes. Then, the transcript abundance of each gene was calculated as TPM in each sample. The Pearson correlation coefficient of the gene expression level of two biological repeats was calculated separately in the three treatment groups of M009, M350 and M374. In the roots of M009, M350 and M374, the r values were 0.83, 0.74 and 0.83, respectively ( Figure S3d-f). The r values of M009, M350 and M374 leaves were 0.93, 0.77 and 0.40, respectively ( Figure S3a-c). These results indicated that our datasets had good biological repeatability and were suitable for subsequent analysis except for the M374 leaves dataset.

| Differential gene expression in response to salt treatment
To identify the DEGs in M009, M350 and M374 under salt treatment, FDR value <0.01 and |log 2FC| ≥ 1 were set as thresholds under salt treatment, there were 3067 and 21,426 DEGs in the leaves and roots of M009, respectively (Figure 1a,d). Among them, there were 1596 upregulated genes and 1471 downregulated genes in leaves, as well as there were 11,093 upregulated genes and 10,333 downregulated genes in roots. Furthermore, 7031 and 2947 DEGs in the leaves and roots of M350 were also identified, respectively (Figure 1b,e). Among them, 2602 genes were upregulated and 4429 genes were downregulated in leaves, and 1889 genes were upregulated and 1058 genes were downregulated in roots. In addition, there were 2507 DEGs in the roots of M374, including 824 upregulated genes and 1683 downregulated genes (Figure 1f). Overall, the number of DEGs in M350 and M374 was much lower than that in M009 under salt treatment. Volcano plots were used to show the fold change of DEGs in leaves (Figure 1a-c) and roots (Figure 1d-f) of the three species, and further revealed that the number of DEGs in M009 was greater than that in M350 and M374. lowest p values were analysed. In all, 11 salt tolerancerelated GO terms were enriched in the roots and leaves of M009, including hydrogen peroxide metabolic process, hydrogen peroxide catabolic process, reactive oxygen species metabolic process, peroxidase activity, oxidoreductase activity, antioxidant activity, glutathione transferase activity, response to water deprivation, auxin-activated signalling pathway, ABA metabolic process and oxidoreductase activity, and acting on single donors with incorporation of molecular oxygen ( Figure S4a,c). Three salt tolerance-related GO terms were enriched in the roots and leaves of M350, including regulation of hormone levels, flavonoid metabolic process and hormone biosynthetic process ( Figure S4b,d). The GO term associated with salt tolerance was not enriched in M374 roots ( Figure S4e). The number of salt tolerance-related GO terms enriched in the roots and leaves of M350 and M374 was less than that in the roots and leaves of M009, indicating that M009 is more sensitive to salt stress than M350 and M374.

| Enriched differential metabolic pathways in response to salt stress
To study the molecular mechanism of the different salt tolerances among M009, M350 and M374, KEGG enrichment was used to analyse the metabolic pathways associated with salt tolerance. In the roots or leaves of M009, the pathways for upregulated genes were mostly enriched in phenylpropanoid biosynthesis, glutathione metabolism, plant hormone signal transduction, calcium signalling pathway and betalain biosynthesis ( Figure S5a,c). The most enriched pathways of the upregulated genes in the roots or leaves of M350 included sesquiterpenoid and triterpenoid biosynthesis, nicotinate and nicotinamide metabolism, plant hormone signal transduction, the MAPK signalling pathway, monoterpenoid biosynthesis, phenylpropanoid biosynthesis, and arginine and proline metabolism ( Figure S5b,d). The upregulated genes in the roots of M374 were mostly enriched in the pathways of flavonoid biosynthesis and valine, leucine and isoleucine degradation ( Figure S5e). These enriched metabolic pathways are mainly related to scavenging of reactive oxygen species, biosynthesis of osmotic protectants and signal transduction of plant hormones, which may help us elucidate the strategy of how to adapt to high-salt conditions in Miscanthus.

| Analysis of orthologous DEGs in response to salt stress
The amino acid sequences of three reference transcriptomes corresponding to M009, M350 and M374 were aligned to obtain 10,721 groups of orthologous transcripts. The types of orthologous transcripts among M009, M350 and M374 included one-oneone, one-one-multiple, multiple-multiple-multiple, multiple-multiple-one, etc. (Table S1). Among the 2698 upregulated transcripts with orthologs in the roots of M009, most of their corresponding orthologs in M350 and M374 were highly and steadily expressed in the roots of both the control and treatment groups (Figure 2a). Among the upregulated transcripts in the roots of M350 (433) and M374 (199), the gene expression levels of most of their corresponding orthologs in the roots of M009 were upregulated (Figure 2b,c). Furthermore, among the 593 upregulated transcripts in the leaves of M009, the expression levels of most corresponding orthologs in the leaves of M350 were not evidently changed under salt treatment ( Figure S6a). Similarly, among 1121 upregulated transcripts in leaves of M350, the expression levels of most corresponding orthologs in M009 leaves were not evidently changed ( Figure S6b). These results indicated that the transcriptional variations in M009 under salt stress were greater than those in M350 and M374. Many of the transcriptional variations in M009 were only passive or involuntary responses to salt stress. Among 10,721 groups of orthologous transcripts in M009, M350 and M374, there were 205 genes involved in hormone signal transduction (Figure 3a), the MAPK signalling pathway (Figure 3b), phenylpropanoid biosynthesis (Figure 3c), arginine and proline metabolism ( Figure 3d) and glutathione metabolism (Figure 3e) in the roots of M009, M350 and M374, and these pathways have been demonstrated to play an important role in responding to salt stress. Among 205 genes, the expression profiles of 117 genes were upregulated under salt treatment of M009, while their corresponding orthologs in M350 and M374 were highly and steadily expressed in both the control and treatment groups (Figure 3). Notably, the expression levels of 117 salt-related genes in M350 and M374 roots were also generally higher than those in M009 roots even under control growth conditions. These results indicated that M350 and M374 might acquire strong salt tolerance by maintaining high expression levels of 117 salt tolerance-related genes under both control conditions and salt treatment conditions. In contrast, M009 might alleviate salt stress by inducing the expression of 117 salt tolerant-related genes under salt treatment. Furthermore, many genes which were identified according to the expression patterns of 117 genes might play an important role in the salt tolerance of Miscanthus.

| Expression pattern validation of candidate genes
The DEGs screened from the analysis of the expression profile were further verified using qRT-PCR. Six known genes of salt tolerance including delta-1-pyrroline-5carboxylate synthase (P5CS), glutathione S-transferase (GST), aquaporin (NIP and TIP), jasmonate-induced protein (JIP), calmodulin (CaM) and a histone gene (H2A.W), which might be a potential salt tolerance gene, were selected for qRT-PCR validation. These seven genes were induced in M009 and constitutively highly expressed in M350 and M374 in the RNA-seq data (Figure 4a). The qRT-PCR experimental results of these seven genes were basically consistent with the expression pattern of these genes in the RNA-seq data, which showed that the RNAseq data were reliable and that these genes responded to salt stress in Miscanthus. In the meantime, H2A.W had the same expression patterns as GST, P5CS, NIP, TIP, JIP and CaM in Miscanthus under salt stress, which indicates that H2A.W might take part in plant resistance to salt stress.

| Identification and sequence analysis of MsaH2A.W
The full-length cDNA of MsaH2A.W was 886 bp including a 480 bp open reading frame, encoding 159 amino acids ( Figure 5a) with a 16.57-kDa molecular mass and 10.68 isoelectric point. The amino acid sequence of MsaH2A.W as a probe was used to search for homologous sequences in other species through the NR database of the BLAST program. The amino acid sequences of all H2A.W genes were aligned and consequently showed that the amino acid sequences of all H2A.W genes were highly conserved in different species and contained a specific KSPKK motif at the C-terminus (Figure 5c). According to phylogenetic analysis, the evolutionary relationship of H2A.W in different species showed that MsaH2A.W had the closest evolutionary relationship with SbH2A.4 (Figure 5b).

| Overexpression of MsaH2A.W enhanced salt stress tolerance
To further verify whether MsaH2A.W responded to salt stress, we generated transgenic Arabidopsis lines that overexpressed MsaH2A.W with the CaMV 35S promoter. The relative expression level of MsaH2A.W in transgenic Arabidopsis lines was analysed through semiquantitative RT-PCR, and consequently showed that OE2 and OE7 transgenic Arabidopsis lines had the highest transcription levels ( Figure 6a). Therefore, OE2 and OE7, two independent homozygous transgenic lines, were obtained and further characterized (Figure 6a). In a control growth medium, there were no visible differences in root length between the WT and OE lines. However, under salt treatment, the primary root length of OE2 and OE7 was significantly longer than that of WT (Figure 6b). OE2 and OE7 exhibited ~1.5 times longer roots than WT (Figure 6c). These results were clearly indicated that overexpression of MsaH2A.W increased the salt tolerance of transgenic Arabidopsis plants and this gene could play critical physiological roles in the salt stress response.

| DISCUSSION
Soil salinization is the most serious environmental threat to crop yield and quality improvement (Munns & Gilliham, 2015). Here, we used RNA-Seq technology to generate the de novo assembled transcriptomes of M009, M350 and M374. By comparing the expression patterns of salt tolerance-related genes in these three species under salt stress, we evaluated the genetic basis of these three species in response to salt stress and identified candidate genes that might be used for improving salt tolerance in plants.
Plants develop a variety of countermeasures including physiological, biochemical and molecular processes, handle adverse salt stress conditions (Van et al., 2020). The interaction of different molecular, cellular, metabolic and physiological mechanisms determines plant resistance to salt stress (Gupta & Huang, 2014). In our study, M350 and M374 were more tolerant to salt stress than M009 by observing the growth status, leaf wilt and chlorosis degree of the three species under salt treatment ( Figure S1). Heterosis is ubiquitous in major crops, such as maize, wheat and rice (Schwarzwälder et al., 2022;Xiao et al., 2021;Zhou et al., 2021). The hybrid generation was obtained by crossing M009 and M374. The insensitivity of their hybrid to salt stress might be partly caused by heterosis, and the other part was estimated to be from the salt resistance genes in M374. Furthermore, the number of DEGs in M009 was much greater than that in M009, M350 and M374 in response to salt stress (Figure 1), which indicated that M009 was more severely influenced by salt stress than M350 and M374. The top 20 GO terms enriched in upregulated genes were analysed in M009, M350 and M374. The number of salt tolerancerelated GO terms significantly enriched in the upregulated transcripts of roots and leaves of M350 and M374 was less than that in M009 ( Figure S4), indicating that M009 was more sensitive to salt stress than M350 and M374. This result was consistent with its salt-sensitive phenotype. Moreover, most of the upregulated genes F I G U R E 3 Heatmap of salt tolerance-related genes in the roots of M009, M350 and M374. (a) Hormone signal transduction; (b) MAPK singalling pathway; (c) phenylpropanoid biosynthesis; (d) glutathione metabolism; (e) arginine and proline metabolism. Red and blue colours represent upregulated and downregulated transcripts, respectively. RC and RT represent the roots of the control and salt treatment groups, respectively.
were significantly enriched in the scavenging of reactive oxygen species, biosynthesis of osmotic protectants and signal transduction of plant hormone-related pathways ( Figure S5). These pathways have been verified to be associated with salt stress tolerance in multiple plants Silva-Ortega et al., 2008;Yu et al., 2020;Zhao et al., 2021). This result indicated that these GO terms and KEGG pathways played an important role in the Miscanthus response to salt stress.
We further analysed the expression profiles of orthologous transcripts of M009, M350 and M374 under salt treatment. Most of the orthologs of the upregulated genes in the roots of M009 under salt stress showed no significant or small differences in the expression F I G U R E 4 Confirmation of the RNA-seq data with qRT-PCR. (a) Expression pattern of seven salt tolerance-related genes through RNAseq. Red and blue colours represent upregulated and downregulated transcripts, respectively. RC and RT represent the roots of the control and salt treatment groups, respectively. (b) Expression pattern of seven salt tolerance-related genes through qRT-PCR. *p < 0.05, **p < 0.01, ***p < 0.001. levels before and after salt stress in M350 and M374 ( Figure 2a). However, their expression levels in M350 and M374 were much higher than those in M009 under either normal growth or salt stress conditions. This result showed that these genes were induced in the roots of M009 and constitutively highly expressed in the roots of M350 and M374 under salt stress. In leaves, the orthologous transcripts of M009 and M350 had no specific expression pattern under salt stress. Previous studies have shown that roots are more sensitive to abiotic stress than leaves in plants (Group, 2009;Yang et al., 2017). In our study, the DEGs and the number of salt-tolerant GO terms enriched in the roots of the three species under salt stress were greater than those in the leaves (Figure 1 and Figure S4). Therefore, these results implied that roots were the main tissue responding to salt stress, and we focused on DEGs in roots for further analysis. When plants are subjected to salt stress, stress signals are perceived by receptors and many secondary signal molecules are produced such as ROS, Ca 2+ and plant hormones, which activate a series of related genes and improve salt stress resistance (Quan et al., 2007;Zhang & Shi, 2013;Zhu, 2002). Then, a total of 205 orthologous genes related to five salt-tolerant pathways, including hormone signal transduction, the MAPK signal pathway, phenylpropanoid biosynthesis, arginine and proline metabolism, and glutathione metabolism (Chen et al., 2013;Cheng et al., 2021;Mehlmer et al., 2010), were screened from the roots of orthologous transcripts, and their expression profiles were further analysed. Among them, 117 genes were induced in M009 and constitutively highly expressed in M350 and M374 under salt treatment (Figure 3). These results illustrated that M350 and M374 could pre-adapt to salt stress by constitutively high expression of a variety of salt tolerance genes, while M009 alleviated salt stress by inducing the expression of some salt tolerance-related genes.
Six known salt tolerance genes, P5CS (Silva-Ortega et al., 2008), GST (Hao et al., 2021), NIP , TIP (Wang, Li, et al., 2011), JIP (Ali & Baek, 2020) and CaM (Guan et al., 2020), were further selected for qRT-PCR validation (Figure 4). The induction of P5CS, a key enzyme of the proline biosynthetic pathway, could improve proline accumulation, and proline, as an osmotic protection agent, could enhance the salt tolerance of transgenic plants under salt stress (Chen et al., 2013;Funck et al., 2020;Silva-Ortega et al., 2008). GST, as a strong non-enzymatic antioxidant, has been reported to be involved in the abiotic stress response, which could alleviate the damage of salt stress to plants by reducing intracellular ROS levels . Both TIPs and NIPs are water channel protein family genes that improve the salt resistance of plants by regulating water and osmotic balance Zhang et al., 2020). JAs play an important role in plant resistance to abiotic stresses by promoting proline synthesis, inducing specific gene expression and synthesizing large amounts of JIPs (Ali & Baek, 2020). CaM is a major receptor for Ca 2+ concentration fluctuations and plays a key regulatory role in the salt resistance response of plants by regulating hormone signalling, ion transport and gene transcription (Li et al., 2022;Zhang, Huang, et al., 2021). We also analysed the expression levels of predicted salt-tolerant genes, and a histone variant gene with the same expression pattern as these six known salttolerant genes was screened. We speculated that this histone variant gene might be related to plant salt tolerance. The qRT-PCR experimental results of seven genes were basically consistent with the expression pattern of these genes in the RNA-seq data (Figure 4), which meant that the RNA-seq data were accurate and reliable. Based on the C-terminal conserved motifs, H2A variants are classified into three types, including H2A.Z, H2A.X and H2A.W (Talbert et al., 2012). H2A.Z WT, OE2 and OE7 seedlings grown on 1/4 PNS medium with or without 100 mM NaCl for 10 days. (c) Primary root length of WT, OE2 and OE7 with or without 100 mM NaCl for 10 days. WT, wild-type. **p < 0.01. Scale bar: 1 cm. is only ~60% similar to canonical H2A in the same species, and the C-terminal tail of H2A.Z is shorter than that of H2A (Zlatanova & Thakar, 2008). H2A.X variant contains a conserved SQEF motif at its C-terminus (Bönisch & Hake, 2012). H2A.W variants contain an extended C-terminal tail with a conserved KSPKK motif (Yelagandula et al., 2014). In our study, the amino acid sequence of the histone variant gene was highly conserved and contained a specific KSPKK motif at the C-terminus (Figure 5), indicating that this histone variant gene was a H2A.W variant. At present, a few histone variants have been confirmed to be involved in salt stress (Nguyen & Cheong, 2018;Qiu et al., 2006). Under salt stress, H2A.Z-containing nucleosomes are evicted from the AtMYB44 promoter region to release the repressors of AtMYB44 and then activate AtMYB44 gene transcription in responds to salt stress (Nguyen & Cheong, 2018). In Arabidopsis, the linker histone variant HIS1-3 responds to salt stress by acting upstream of the SOS pathway to regulate SOS1 and SOS3 gene expression (Wu et al., 2022). TaH2A.7 gene as an H2A.W variant could improve the drought tolerance of transgenic Arabidopsis by reducing water loss and promoting stomatal closure (Xu et al., 2016). However, the histone variant H2A.W has not been reported to respond to salt stress. Therefore, we further explored whether H2A.W was related to the salt tolerance of plants. We cloned the histone variant MsaH2A.W coding sequence from M350 and consequently obtained transgenic Arabidopsis plants with MsaH2A.W to prove whether MsaH2A.W could enhance the salt stress tolerance of transgenic Arabidopsis plants. In this experiment, we found that the primary root lengths of OE2 and OE7 were significantly longer than that of WT after 10 days with 100 mM NaCl treatment ( Figure 6). This result indicated that overexpression of MsaH2A.W could enhance the salt tolerance of transgenic Arabidopsis plants.
There are complex regulatory mechanisms in plants for responding to salt stress, including physiological, morphological, biochemical and molecular changes . Under salt stress, the genes that regulate these processes play an important role in the improvement of salt-tolerant crop varieties Ziska et al., 2012). In our study, according to the expression pattern of known salt tolerance genes, we identified a histone variant, MsaH2A.W of M350, which improved salt tolerance in transgenic Arabidopsis plants. Our research initially revealed the underlying genetic basis of salt stress resistance in Miscanthus, which is of great importance for cultivating salt-resistant species using salt resistance gene resources of Miscanthus. Furthermore, MsaH2A.W has great potential as a salt tolerance gene in Miscanthus and its molecular mechanism will be identified in future work.