<i>MsaH2A.W</i> is identified response to salt tolerance in <i>Miscanthus sacchariflorus</i>

Miscanthus is a perennial forage plant with great potential for high stress tolerance and biomass yield. It has strong adaptability growing in saline land avoids competition grain crops arable lands. However, little known about the underlying genetic basis of adaptation to salt stress. Two diploid species genus Miscanthus, sinensis sacchariflorus, were focus this study. The transcriptome variations these varieties their hybrid analysed using RNA-seq technology under treatment. number differentially expressed genes M. was much higher than that sacchariflorus stress, which indicated require less transcriptional variation. In addition, most salt-tolerant enriched pathways induced roots constitutively highly According expression pattern genes, histone variant gene MsaH2A.W mined consequently proved first time it could enhance transgenic Arabidopsis plants. Overall, study provides valuable resources studying resistance Miscanthus. Identification can promote improvement molecular breeding salt-resistant species. 芒属植物是多年生饲草植物, 因具有抗逆性强和生物质产量高等特点, 有着较大的开发潜力及应用价值。它在非生物胁迫环境下具有一定程度的耐受性, 可以利用盐碱地等各种边缘土地上进行种植, 避免与粮争地。然而, 芒属植物适应盐胁迫的遗传基础尚不清楚。本研究采用二倍体的芒、荻及其在杂交种为研究材料。利用RNA-seq技术分析了盐处理下芒、荻及其杂交种的转录组变化。结果表明, 在盐胁迫下, 芒中的差异表达基因数量远高于荻及杂交种, 结果表明荻及杂交种需要较少的转录变化来应对盐胁迫。此外, 富集到耐盐途径中的大部分耐盐基因在芒根中诱导表达, 在荻及杂交种根中组成型高表达。根据这些已知耐盐基因的表达模式, 组蛋白变体基因MsaH2A.W被挖掘并首次证明了它能提高转基因拟南芥的耐盐性。总之, 本研究为研究芒属植物应对盐胁迫的遗传基础提供了宝贵的遗传资源, 并为耐盐品种的遗传改良和分子育种奠定了基础。 Salt major abiotic factor threatens agricultural production worldwide (Hazell & Wood, 2008). Approximately 45 million hectares world's are affected by salinity (Shrivastava Kumar, 2015). High intensity long periods change physiological processes morphological structure plants, stunts growth development plants may even lead death (Paul, 2013). To maintain basic life activities alter specific cellular biochemical activating large accumulating corresponding proteins (Van et al., 2020; Zhao 2021). Therefore, importance comprehensively mechanism resist external environment, activate diverse physiological, mechanisms activity (Gong, 2021; Zhu, 2002). Antioxidant enzymes non-enzymatic compounds play key role ROS detoxification (Bohnert Sheveleva, 1998; Gupta Huang, 2014). antioxidant enzyme effectively (Roxas 2000; Xu 2015; Yang Based on osmotic signalling pathway, efficiently alleviate regulating expression, osmolytes (proline, sugar polyol) water transport systems (Shumilina 2019). interaction several phytohormones, such as abscisic acid (ABA), auxins (indole acetic acid), jasmonic (JA), cytokinins, salicylic brassinosteroids, participate response regulate tolerance-related transmitting signals (Ma 2006; Osthoff stress-activated transcription factors (TFs), WRKY NAC (NAM, ATAF1/2 CUC2), crucial regulatory pathway stress-related together other (Gao Li Shinozaki Yamaguchi-Shinozaki, 1997). At present, some research ROS, hormone TFs advanced multiple very limited. C4 grass, belongs Saccharinae subtribe Andropogoneae tribe (Poaceae) (Paterson, 2012). subtribe, Saccharum Sorghum have been extensively researched beneficial characteristics bioenergy production. Compared sorghum sugarcane, possesses cellulose/hemicellulose per unit area (Zhang, Ge, There differences lignocellulose components stems 179 indicates China rich diversity (Xu 2020). good paper-making properties (Miao 2021), be used high-quality feed raw material edible fungi (Finet important economic value ecological significance an ornamental (Mitros cultivated increasing meet demand (Heaton Hence, widely applied (Ge 2019; 2014; Sun genomes genera well described, floridulus lutarioriparius 2021) These genome data helpful functional genomic date, mainly focused utilization biomass, (Bukowski Dai 2022; Golfier 2017; Lee Kuan, wild grow marginal avoid competing lands (Fu 2009; Wang, Shao, 2011). Studies shown relatively rate accumulation compared (Plazek Stavridou 2017). moderately crop, acquired wide range stress-responsive during long-term (Hung Ogura Yura, Chen al. (2017) showed 70 genotypes had broad tolerance, different materials (Chen By comparative transcriptomic analysis five populations 59 shared found respond high-salinity environments, approximately 70% them proven associated responses (Song MINAC9, MINAC10 MINAC12, improve (He 2018; 2016). only few studies performed germplasm not deeply studied. eukaryotes, histones chromatin structure. Several kinds histones, including H2A, H2B, H3 H4, interact constitute nucleosome octamer core (Kornberg, 1974; Histone variants integrity, chromosome segregation (Yelagandula altered or levels (Nguyen Cheong, Talbert Henikoff, 2010). linker HIS1-3 drought (Ascenzi Gantt, 1999; Wu 2022). Under H1-S tomato obtained antisense strategy, stomatal closure (Scippa 2004). Similarly, TaH2A.7 wheat also promoting H2A.Z participates environmental temperature perception nutrient (Kumar Wigge, 2010; Smith H3.2 RH3.2A rice involved (Qiu 2006). Although H2A.Z, H2A.7 H3.2A whether there more elucidated (Chang Zhu Here, transcriptomes sinensis, treatment analysed. strategies patterns three investigated, explored. Then, based we identified MsaH2A.W, its overexpression enhanced This researchers related lays cultivation new varieties. (M009) (M350) collected from Hunan Gansu, China, respectively. Both (2n = 2× 38). M009 M350 transplanted into resource nursery Agricultural Experiment Station Shandong University, (M374) crossing M350. M374 grew same location. rhizomes M009, separated single buds, each bud transferred plastic pot perlite grown greenhouse 1/2 Hoagland solution, 16-h light/8-h dark cycle at mean 26°C ± 1°C relative humidity 50%. When seedlings three-leaf-age, salt-treatment groups cultured solution (200 mM NaCl), control irrigated before. After 12 h treatment, leaves RNA-seq. biological replicates set up M374. phenotypes after 28 days TRIzol reagent (Invitrogen) RNA isolation all samples. DNaseI eliminate DNA contamination concentration quality sample measured Nanodrop 2000c spectrophotometers 0.8% agarose gel electrophoresis. custom high-throughput Illumina library method followed prepare libraries. libraries Qubit 2.0 Fluorometer (Life Technologies), Agilent Bioanalyzer 2100 system 3% 2 × 150 bp paired-end configuration sequencing cDNA libraries, lengths fragments 300–500 bp. HiSeq 4000 adopted carrying out sequencing. Trimmomatic initially filtering reads removing adapter-containing reads, low-quality empty (Bolger clean two repeats combined groups. Trinity assemble de novo (Grabherr RSEM accurately quantify transcripts (Li Dewey, 2011) calculate kilobase (TPM) represent transcript abundance unigene. Sequences TPM values one filtered assembled transcripts. cd-hit software remove redundant sequences similarity greater 90%. Finally, reference separately. mapping transcriptome. comparing between groups, (DEGs) screened differential criteria (|log2FC | ≥ 1 FDR <0.01). obtain annotation transcript, unigenes aligned UniProt database (http://www.uniprot.org/) means local BLAST. GO annotations KO loading alignment results Trinotate sqlite database. clusterProfiler package R identify terms annotated series p < 0.01 KEGG DEGs. metabolic 0.05 amino orthologous proteinortho6.pl software. analysing DEGs strategy environment elucidated. candidate identified. verify reliability data, six P5CS, GST, NIP, TIP, JIP CaM, excavated gene, H2A.W chosen qRT-PCR analysis. Total root isolated reagent. synthesized Evo M-MLVPlus Synthesis Kit (Accurate Biology). manufacturer's instructions, Bio-Rad CFX96 Real-Time instrument UltraSYBR Mixture (Low ROX) seven normalized those internal control. Three gene. Gene primers listed Table S2. sequence extracted data. AtH2A.W.6 (NM_124845.4) Arabidopsis, EsH2A.6 (XM_006401487.2) Eutrema salsugineum, SbH2A.4 (XP_002441223.1) bicolor, OsH2A.4 (XP_015640099.1) Oryza sativa Japonica Group, ZmH2A (ACG38394.1) Zea mays, SiH2A.4 (XP_004961835.1) Setaria italica, MuH2A.2 (XP_009409009.1) Musa acuminata subsp. malaccensis downloaded NR BLAST program according MsaH2A.W. DNAMAN phylogenetic coding cloned pCAMBIA3300-3flag vector generate 35S::MsaH2A.W-3flag constructs. Agrobacterium tumefaciens GV3101 infect floral dip. Overexpression wild-type (WT) (ecotype Col-0). Transgenic MS medium 0.005% Basta. independent lines (OE2 OE7) confirmed semiquantitative RT-PCR selected phenotypic Primers S3. investigate sterile seeds WT, OE2 OE7 sown 1/4 (PNS; Gong 2004) solid medium. 5 vernalization 4°C, vertically 22°C cycle. Five-day-old PNS without 100 NaCl sensitivity Phenotypic 10 days. Furthermore, entire primary length ImageJ software, statistical WT experiments. well-cultured treated 200 leaf wilting degree M374, clearly better Moreover, substantial chlorosis observed (Figure S1). observations stronger M009. tolerances through next-generation (NGS). assembly. A total 71,719, 76,882 61,879 N50 1469, 1406 1459 bp, GC content 52.48%, 52.10% 52.56% respectively (Table 1). mapped unigenes, average ratios 91.10%, 90.57% 89.37%, distribution unigene assembly 46.03%, 42.32% 49.13% over 600 S2). suggested eligible transcriptomes. calculated sample. Pearson correlation coefficient level separately r 0.83, 0.74 S3d–f). 0.93, 0.77 0.40, S3a–c). our datasets repeatability suitable subsequent except dataset. <0.01 |log 2FC| thresholds 3067 21,426 1a,d). Among them, 1596 upregulated 1471 downregulated leaves, 11,093 10,333 roots. 7031 2947 identified, 1b,e). 2602 4429 1889 1058 2507 824 1683 1f). lower Volcano plots show fold 1a–c) 1d–f) species, further revealed classify categories DEGs, term enrichment top 20 lowest all, 11 hydrogen peroxide process, catabolic reactive oxygen peroxidase activity, oxidoreductase glutathione transferase deprivation, auxin-activated ABA process acting donors incorporation S4a,c). M350, regulation levels, flavonoid biosynthetic S4b,d). S4e). indicating sensitive among analyse tolerance. mostly phenylpropanoid biosynthesis, metabolism, signal transduction, calcium betalain biosynthesis S5a,c). included sesquiterpenoid triterpenoid nicotinate nicotinamide MAPK monoterpenoid arginine proline metabolism S5b,d). valine, leucine isoleucine degradation S5e). scavenging protectants transduction hormones, help us elucidate how adapt high-salt conditions 10,721 types one–one–one, one–one–multiple, multiple–multiple–multiple, multiple–multiple–one, etc. 2698 orthologs steadily both 2a). (433) (199), 2b,c). 593 evidently changed S6a). 1121 S6b). Many passive involuntary 205 3a), 3b), 3c), 3d) 3e) demonstrated responding profiles 117 while 3). Notably, salt-related generally conditions. might acquire maintaining contrast, inducing tolerant-related many profile verified qRT-PCR. Six delta-1-pyrroline-5-carboxylate synthase (P5CS), S-transferase (GST), aquaporin (NIP TIP), jasmonate-induced protein (JIP), calmodulin (CaM) (H2A.W), validation. 4a). experimental basically consistent reliable responded meantime, CaM take part full-length 886 480 open reading frame, encoding 159 acids 5a) 16.57-kDa mass 10.68 isoelectric point. probe search homologous program. conserved contained KSPKK motif C-terminus 5c). analysis, evolutionary relationship closest 5b). generated overexpressed CaMV 35S promoter. semi-quantitative RT-PCR, highest 6a). OE7, homozygous lines, characterized medium, no visible OE lines. significantly longer 6b). exhibited ~1.5 times 6c). increased critical roles response. Soil salinization serious threat crop yield (Munns Gilliham, RNA-Seq evaluated improving Plants develop variety countermeasures processes, handle adverse molecular, cellular, determines (Gupta study, tolerant observing status, wilt Heterosis ubiquitous crops, maize, (Schwarzwälder Xiao Zhou generation insensitivity partly caused heterosis, estimated 1), severely influenced S4), result salt-sensitive phenotype. hormone-related S5). (Cheng Silva-Ortega 2008; Yu played We Most significant small before either normal Previous (Group, Figure S4). implied main tissue subjected perceived receptors secondary molecules produced Ca2+ (Quan 2007; Zhang Shi, 2013; pathways, Cheng Mehlmer 2010), transcripts, illustrated pre-adapt alleviated genes. P5CS (Silva-Ortega 2008), GST (Hao NIP (Yan 2014), TIP (Wang, Li, 2011), (Ali Baek, 2020) (Guan 2020), validation 4). induction accumulation, proline, protection agent, Funck antioxidant, reported response, damage reducing intracellular (Yang TIPs NIPs channel family balance JAs stresses synthesis, synthesizing amounts JIPs receptor fluctuations plays signalling, ion Zhang, predicted screened. speculated 4), meant accurate reliable. C-terminal motifs, H2A classified types, H2A.X (Talbert ~60% similar canonical tail shorter (Zlatanova Thakar, contains SQEF (Bönisch Hake, contain extended 5), variant. Qiu H2A.Z-containing nucleosomes evicted AtMYB44 promoter region release repressors then responds 2018). upstream SOS SOS1 SOS3 (Wu loss explored prove experiment, 6). complex morphological, changes (Zhao (Godoy Ziska variant, improved Our cultivating will future work. Cuixia designed supervised project; Ting Yu, Senan Xinwei Hou data; Yancui Pingping Xu, Cheng, Xitong Guofeng Geng, Zhiqiang Pan, Shukai Dusheng Lu, Shubo Gu, Zhixin Liu carried wrote manuscript; revised manuscript. supported National Key R&D Program (grant no. 2022YFD1201700), Variety Improvement Project Province, C.C. 2017LZN028) Natural Science Foundation nos 31871267, 31271352) financial support. authors declare conflicts interests. article NCBI SRA project ID PRJNA908119. Data S1. Please note: publisher responsible functionality any supporting information supplied authors. Any queries (other missing content) should directed author article.