Molecular mechanism of phytohormones regulating dormancy and germination of barley seeds

Molecular mechanism of phytohormones regulating dormancy and germination of barley seeds | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Molecular mechanism of phytohormones regulating dormancy and germination of barley seeds Jun Wu, Bing Zhou, Bo Li, Peng Wang, Ying Zhang, Liya Luo, Yanhao Xu, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6858206/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Under adverse conditions such as continuous rainfall or high humidity, mature grains are prone to pre-harvest sprouting (PHS) on the parent plant, significantly impacting crop yield and quality. While abscisic acid (ABA) and gibberellin (GA) are well-known regulators of seed germination, the molecular mechanisms by which other phytohormones influence this process remain underexplored. A germination test of 236 barley varieties showed a germination index range of 5.22 to 97.17, indicating extensive genetic diversity. Hormone treatments on fast-germinating Harrington and slow-germinating Stirling revealed that ABA, cytokinin, and salicylic acid inhibited germination, whereas GA and ethylene promoted it. Endogenous hormone quantification indicated that variations in PHS resistance between Harrington and Stirling were primarily due to differences in ABA, GA, auxin, and cytokinin levels. Transcriptomic analysis of barley embryonic tissues identified 260 hormone-responsive differentially expressed genes, with 54 of these genes showing specificity in their expression during germination. Comparative resequencing data from two pairs barley varieties exhibiting contrasting germination traits revealed 19 genes with substantial genetic variation in their coding regions, implicating these genes in the regulation of barley germination properties. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. INTRODUCTION Dormancy, defined as the incapacity of an intact viable seed to complete germination under favourable conditions. Weak seed dormancy in barley leads to pre-harvest sprouting (PHS), but strong dormancy results in poor malting quality, which causes significant economic loss worldwide cereal-growing regions (Tai et al., 2021 ). PHS occurs in almost every cereal-growing region globally and is responsible for up to $ 1 billion in annual losses (Miao et al., 2013 ; Lang et al., 2021 ; Vetch et al., 2019 ). Seed dormancy and germination are crucial traits in cereals, as seeds lacking dormancy are highly susceptible to PHS (Bonnardeaux et al., 2008 ). Domestication and modern breeding practices aim to promote rapid and uniform plant establishment but often reduce grain dormancy, making crops more susceptible to PHS (Newberry et al., 2018 ). Phytohormones such as gibberellin (GA), ethylene (ETH), brassinolide (BR), and cytokinin (CK) promote seed germination and positively regulate PHS, while abscisic acid (ABA), auxin, salicylic acid (SA), and jasmonic acid (JA) negatively regulate PHS by inhibiting germination (Yan et al., 2021 ). The application of GA enhances seed germination and alleviates dormancy (Nonogaki, 2014 ; 2017 ). For example, the transcriptional abundance of genes such as KO1 , KAO1 , GA20ox , GA3ox , and GID1b increases during GA synthesis in barley germination (Betts et al., 2020 ). ETH similarly promotes germination and dormancy release, as exogenous ETH or ethephon can break both primary and secondary seed dormancy (Li et al., 2019 ). Other hormones, such as BR and CK, also play critical roles in regulating germination through signaling pathways (Xiong et al., 2022 ; Yuan et al., 2008 ). ABA levels decrease during seed imbibition, facilitating germination in both barley and Arabidopsis (Jacobsen et al., 2002 ; Millar et al., 2006 ). Auxin enhances ABA-induced dormancy by interacting with ARF (auxin response factor) transcription factors to maintain ABI3 and ABI5 expression during seed imbibition (Liu et al., 2013 ). Similarly, SA and JA regulate germination under stress conditions (Li et al., 2017 ; Ju et al., 2019). Barley ( Hordeum vulgare L.) is an ancient crop that ranks fourth globally in terms of cultivated area and total production (Zeng et al., 2018 ). Understanding the molecular mechanisms underlying phytohormone regulation of barley seed dormancy and germination is critical to fine-tune PHS and rapid germination. We need to create an ideotype with an adequate dormancy level to prevent PHS but capable of rapid and efficient germination under the right conditions shortly after harvest; this will provide high malting quality. We propose that fine-tuning the genes involved in grain dormancy, seed germination, and malting quality will likely achieve the outcome. This study used two barley varieties, the PHS-sensitive Harrington and the PHS-resistant Stirling, to investigate the transcriptional and physiological changes during germination. By analyzing phytohormone regulation and gene expression during germination, the findings of this study provide important insights into the molecular basis of PHS regulation and offer valuable information for breeding PHS-resistant barley varieties suitable for high rainfall and humidity conditions. 2. MATERIALS AND METHODS 2.1 Characterization of germination in different genotypes of barley Different barley varieties were kindly provided by Prof. Meixue Zhou from University of Tasmania. Uniform barley seeds were sterilized with 75% ethanol for 1 minute, followed by 2% sodium hypochlorite for 15 minutes, washed with sterile water, and placed in 9 cm petri dishes with two layers of filter paper. A total of 100 seeds were placed per dish, and 4 mL of distilled water were added to each petri dish, and samples were incubated in the dark at 20 ± 0.5°C. Germination rate (GR) was measured at 24 h, 48 h and 72 h, and germination index (GI) were measured at 72 h. Germination index (GI)=∑(Gt/Dt), Gt is the number of germinated seeds day by day, and Dt is the number of days to germination for the corresponding DG, counted up to day 3. 2.2 Exogenous hormone treatment of Harrington and Stirling Four milliliters of hormone solution at specified concentrations (Table S2 ) were added to each petri dish, with distilled water serving as a control. Dishes were incubated in the dark at 20 ± 0.5°C. The number of germinated seeds (the appearance of white radicle) was recorded at 24 h, 36 h, 48 h, 60 h, and 72 h. 2.3 Measurement of endogenous phytohormones Phytohormone quantification was conducted using an ESI-HPLC-MS/MS (Electrospray ionization-high-performance liquid chromatography-mass spectrometry/mass spectrometry) system equipped with a QTRAP™ 6500 liquid-mass spectrometer (AB Sciex). Full-grain Harrington and Stirling seeds were sampled 24 h and 48 h after incubation. Only seed embryos were collected, immediately frozen on dry ice, and stored in an ultra-low-temperature freezer at − 80°C. Each sample contained 100–300 embryos, for hormone content analysis. Standard solutions of IAA, ABA, JA, SA, GA1, and t Z were prepared in methanol at concentrations ranging from 0.1 ng/mL to 200 ng/mL. The linear standard curves were plotted. 2.4 Expression analysis of phytohormone-related genes Transcriptomic differences in Harrington and Stirling barley were analyzed at 24 h and 48 h of germination using publicly available data (Zhang et al., 2016; accession number GSE66024). Gene expression was quantified as Fragments Per Kilobase of transcript per Million mapped reads (FPKM). Data on Morex barley gene expression during 15 growth stages were obtained to identify germination-specific genes (Mayer et al. 2012 ). Candidate genes involved in GA, ABA, AUX, CTK, ETH, BR, JA, and SA synthesis, metabolism, and signaling were identified from previous reports (Spielmeyer et al., 2004 ; Shu et al., 2016 ; Nguyen et al., 2022 ). Sequences of phytohormone-related genes reported in Arabidopsis thaliana , wheat, rice, and barley were combined with barley genome annotations and GenBank data. A total of 260 phytohormone-related genes were identified through BLAST-based nucleotide and protein homology searches (Mascher et al., 2017 ). Detailed information on these genes is provided in Table S3 . 2.5 Pan-genome sequence analysis Candidate genes identified in the transcriptome analyses were compared using pan-genomic data from four barley varieties (Barke, Morex, Stirling, and Clipper) (Jayakodi et al., 2020 ; Hu et al., 2023 ). Barke and Morex germinated quickly while, the germination index of Stirling and Clipper was below 25, which is highly PHS resistant. 2.6 RNA extraction and real-time fluorescence quantification RNA was extracted using the TRIzol method. RNA quality was assessed using a NanoDrop 2000 UV spectrophotometer and 1% agarose gel electrophoresis. High-quality RNA was stored at − 80°C. cDNA synthesis was performed using the HiScript® II 1st Strand cDNA Synthesis Kit (+ gDNA wiper). Quantitative RT-PCR was conducted on a QuantStudio™ 7 Flex Real-Time PCR System (Applied Biosystems, USA) with a ChamQ SYBR qPCR Master Mix (Vazyme, China). Candidate gene primers were designed using Primer Premier 5 and validated with the NCBI database (Table S4 ). 3. RESULTS 3.1 Germination characteristics of 236 barley germplasm resources In this study, the germination characteristics of 236 barley germplasm resources were assessed using germination rate (24 h-GR24, 48 h-GR48), and germination index (GI). The germination rate and germination index data for the 236 barley varieties are presented in Table S1 . GR24 ranged from 1.00–96.67%, GR48 from 7.67–97.00%, and GI from 5.22 to 97.17. The coefficients of variation for these indices were 82.80%, 40.61%, and 41.24%, respectively, indicating extensive germination traits variation and genetic diversity of this population (Table S5 ). Among the 236 varieties, in 2022 and 2023, the germination index of MSS87, 261, Stirling, Clipper, C2-05-337-2, Irene, 262, Linus, Troon, 260, and USA 8330 − 1679 were below 25 (Table S6 ). Conversely, 12 varieties showed fast germination, such as SHEBA, ETHIOPIA 602, Harrington, Morex, and Barke (Table S7). PHS sensitive Harrington, and PHS tolerant-Stirling were selected for subsequent analyses due to their significant differences in germination traits. At 24 h, Harrington exhibited a germination rate of 62.33%, significantly higher than Stirling (around 1%) (Fig. S1 ). 3.2 Effects of exogenous hormone treatment on germination of Harrington and Stirling Exogenous GA3 significantly increased the germination of Harrington and Stirling with increasing concentrations and ABA inhibited germination (Fig. 1 A, Fig. 1 B). ETH treatment increased Stirling’s germination significantly while Harrington did not response to ETH (Fig. 1 C). IAA and EBR showed dual effects: promoting germination at low concentrations and inhibiting it at higher concentrations (Fig. 1 D and 1 E). JA had no significant impact on Harrington but increased Stirling’s germination (Fig. 1 F). Both 6-BA and SA inhibited the germination of Harrington and Stirling in a dose dependent manner (Fig. 1 G and 1 H). 3.3 Endogenous Hormone Content during Germination Endogenous levels of ABA, GA1, IAA, tZ (trans-zeatin), JA, and SA were measured in Harrington and Stirling embryos at 24 h and 48 h using ESI-HPLC-MS/MS (Fig. 2 ). GA1 levels increased in both varieties during germination, with significantly higher levels in Stirling than Harrington at both time points (Fig. 2 A). ABA level decreased significantly during germination and it was lower in Harrington than Stirling at 24 h (Fig. 2 B). IAA levels also decreased during germination and were consistently higher in Stirling than in Harrington, suggesting a potential role in the observed germination differences (Fig. 2 C). The tZ content increased in both varieties from 24 h to 48 h (Fig. 2 D). JA and SA levels decreased during germination in both varieties (Fig. 2 E and 2 F). 3.4 Identification of Key Differential Expressed Genes (DEGs) in Phytohormone Pathways Using transcriptomic data from Harrington and Stirling at 24 h and 48 h of germination, we chose differential Expressed Genes (DEGs) according to three criterial: firstly, these genes should have roles in phytohormone biosynthesis, metabolism or signaling; secondly, different expression pattern in Harrington and Stirling (fold change ≥ 2); thirdly, these genes were expressed specific at germination stage (4-day embryos, EMB) based on the germination data from 15 developmental stages of Morex (Table S3 ). In the GA pathway, genes such as KAO1 ( ent -kaurenoic acid oxidase), GID1 ( HORVU1Hr1G060810 ), and DELLA ( HORVU4Hr1G006930 ) exhibited higher expression in Stirling than Harrington during germination (Fig. 3 A). In the Morex 4-day embryos (EMB), elevated expression levels of KAO1 ( HORVU7Hr1G003090 ), GA13ox2 ( HORVU4Hr1G050930 ), and GA3ox2 ( HORVU3Hr1G022840 ) were detected. This upregulation may correlate with the observed increase in GA1 content in germinating Harrington and Stirling (Fig. 3 B). Similar trends were observed for ABA-related genes such as NCED (9-cis-epoxycarotenoid dioxygenase, HORVU5Hr1G000320 ), which were upregulated in Stirling (Fig. 4 A). Abscisic aldehyde oxidase (AO) is the last key enzyme in the ABA synthesis pathway, and expression of AO ( HORVU5Hr1G103890 , HORVU7Hr1G114000 , HORVU7Hr1G114020 ) is high in EMB of Morex (Fig. 4 B). PP2C ( HORVU5Hr1G097060 , HORVU3Hr1G085930 ), and PYR1 ( HORVU4Hr1G055220 , HORVU1Hr1G050110 ) in the ABA signaling pathway had higher expression in EMB (Fig. 4 B). The transcript expression of PYR1 and PP2C in the Stirling were significantly higher than in Harrington (Fig. 4 A). The YUCCA family encodes flavin monooxygenase, which is the rate-limiting enzyme in auxin synthesis. The expression of most YUCCA transcripts were up-regulated from 24 h to 48 h during germination, and YUCCA expression were significantly higher in Stirling than in Harrington (Fig. 5 A). YUCCA ( HORVU2Hr1G010060 , HORVU7Hr1G027520 ) were highly expressed during the EMB period in Morex (Fig. 5 B). ARF (auxin response factor, HORVU6Hr1G026730 , HORVU7Hr1G108360 , HORVU7Hr1G051930 ), TIR/AFB ( HORVU1Hr1G021550 , HORVU2Hr1G070800 ) and AUX/IAA ( HORVU1Hr1G086070 , HORVU3Hr1G031460 , HORVU1Hr1G025670 ) were highly expressed during EMB (Fig. 5 B). These eight genes were significantly different between Harrington and Stirling (Fig. 5 A). The expression of GH3 ( HORVU2Hr1G03467 ) and ILR1-like ( HORVU3Hr1G048560, HORVU3Hr1G066450 ) were high during EMB in Morex (Fig. 5 A). Auxin-related genes showed higher expression in Stirling, indicating their involvement in regulating germination (Fig. 5 A). The S-AdoMets ( HORVU6Hr1G063540 , HORVU6Hr1G063490 ), ACS ( HORVU3Hr1G019430 , HORVU3Hr1G019410 ), ACO ( HORVU5Hr1G067530 ), and ETR2 ( HORVU5Hr1G115160 ) accumulated in EMB (Fig. S2 ). Also in Harrington and Stirling, transcript expression of ACS were up-regulated from 24 h to 48 h of germination, and the six ACO were up-regulated from 24 h to 48 h (Fig. 6 A). In BR biosynthesis, signaling and metabolic pathways, the expression of DET2 ( HORVU4Hr1G060850 ) and BR6ox1 ( HORVU2Hr1G002250 ) were significantly higher in Stirling during germination than in Harrington (Fig. S3 A). DWF1 ( HORVU7Hr1G120030 ), BAK1 ( HORVU6Hr1G049080 ), and BES1/BZR1 ( HORVU0Hr1G040070 , HORVU7Hr1G088980 ) were significantly higher in Harrington than in Stirling (Fig. S3 A). Higher expression of DET2 , DWF1 , BR6ox1 , BSU1 , BAK1 and BES1/BZR1 were found during EMB in Morex (Fig. S3 B). The transcript expression of LOG (Lonely Guy, HORVU2Hr1G089620 ), CKX (Cytokinin Oxidase, HORVU3Hr1G019850 ) and ARR (Arabidopsis Response Regulator, HORVU6Hr1G028680 , HORVU3Hr1G114970 ) in CK biosynthesis, signaling and metabolism pathway was significantly higher in Stirling than in Harrington (Fig. S2 C). Among them, HORVU2Hr1G089620 was down-regulated from 24 h to 48 h (Fig. S2 C). The transcript expression of these four genes was higher in expression during the EMB period (Fig. S2 D). JA biosynthesis, signaling, and metabolism-related LOX (Lipoxygenase, HORVU5Hr1G093770 , HORVU6Hr1G000510 , HORVU5Hr1G093700 , and HORVU4Hr1G005920 ), AOS (Allene Oxide Synthase, HORVU4Hr1G066270 , HORVU6Hr1G039440 ), AOC (Allene Oxide Cyclase, HORVU6Hr1G081000 ) and OPR (12-Oxophytodienoic Acid Reductase, HORVU2Hr1G004230 , HORVU7Hr1G036070 ) showed lower expression in Harrington than Stirling (Fig. 6 A). Among them, LOX and AOS had higher transcript expression during EMB (Fig. 6 B). SA biosynthesis includes the isochorismate synthase ( ICS ) pathway and the phenylalanine ammonia-lyase ( PAL ) pathway. The transcript expression of ICS and PAL in both Harrington and Stirling was up-regulated during germination at 24 h and 48 h (Fig. 6 C). ICS ( HORVU5Hr1G057050 ), EPS1 ( HORVU1Hr1G072050 ), PAL2 ( HORVU0Hr1G016330 ), and PAL6-5 ( HORVU2Hr1G089440 ) were highly expressed in barley during the EMB period (Fig. 6 D). The high transcript levels of genes within the ICS and PAL pathways indicate active SA synthesis during the germination, implying a potential regulatory role in the germination process. 3.5 Validation of Differential Gene Expression Eight differentially expressed genes ( HORVU4Hr1G006930 , HORV5Hr1G103890 , HORV7Hr1G027520 , HORV6Hr1G03450 , HORV6Hr1G06390 , HORV4Hr1G060850 , HORV4Hr1G066270 and HORV0Hr1G016330 ) involved in phytohormone synthesis and metabolism were selected for RT-qPCR validation. Results were consistent with transcriptomic data, confirming the reliability of RNA-seq findings. Genes related to AUX, CK, and ABA were identified as key regulators of germination in barley (Fig. S4 ). 3.6 Pan-genome reveals candidate gene variances All of the above genes were induced during germination and they show distinct expression pattern in Harrington and Stirling germination. Accordingly, we conducted a comparative analysis of the 54 genes against the published barley pan-genome to ascertain the presence of any variations within the coding DNA sequence (CDS) region and to explore the potential correlation between these variations and the differential sprouting behaviors observed. Hu et al 2023 . found that significant PAVs (Presence/Absence variants) occurred in genes involved in photoperiod, flowering time through comparative genomics studies of barley in Europe, Australia and North America. Whether barley varieties with different germination rates differ significantly in expression due to PAVs during the long evolution of barley? We compared the presence of PAVs in 54 candidate genes selected from the transcriptome results by four barley varieties in the pan-genomic study: Barke (germination index 88, fast germination), Morex (germination index 85, fast germination), Stirling (germination index 21, slow germination), and Clipper (germination index 23, slow germination). We found that there were only 2 genes with SNPs (single nucleotide polymorphisms) in the CDS region as KAO1 ( HORVU7Hr1G003090 ) and YUCCA ( HORVU2Hr1G010060 ). The NCED ( HORVU5Hr1G000320 ) has multiple base substitutions starting at position 316 bp, and it cause the amino acid sequence change, which may lead to changes in its expression in different barley varieties (Fig. 7 C). There were insertions or deletions of large segments in the CDS region of 17 genes, as shown in Table S8. We found a 3bp insertion and deletion of GID1 , PP2C and BES1 in Stirling and Clipper (Fig. S5 A, B and C). DELLA , TIR1/AFB , ARF , PAL6-5 , and ILR1 all produced large segment deletions (Fig. S5 D, E, F, G, and H). Our results suggest that the 19 candidate genes that are highly or specifically expressed during seed germination have different PAVs, which may lead to genetic differentiation of germination characteristics. These variations likely contribute to differences in germination speed and hormone sensitivity among barley varieties (Table S8; Fig. 7 ) 4. Discussion Barley is a widely cultivated crop with high nutritional value, but PHS significantly contributes to yield losses. Recent studies suggest that phytohormone engineering could mitigate PHS, as genes regulating seed dormancy and germination are significantly enriched in phytohormone metabolic pathways (Nonogaki, 2017 ; Liu et al., 2022 ). Understanding the mechanisms by which phytohormones regulate seed dormancy and germination is essential for developing effective strategies to address PHS. This study aimed to elucidate the molecular mechanisms underlying phytohormone metabolism and signaling during barley seed germination under controlled conditions. To achieve this, we evaluated the effects of exogenous phytohormones on barley germination, quantified endogenous phytohormones using ESI-HPLC-MS/MS, and identified key genes in phytohormone pathways through transcriptomic and pan-genomic analyses. In this study, exogenous GA3 significantly promoted barley germination, while exogenous ABA inhibited it. Endogenous hormone assays revealed that ABA and GA1 levels increased from 24 h to 48 h during germination in both Harrington and Stirling. Stirling had higher ABA and GA1 levels than Harrington, indicating that its endogenous GA1 levels were insufficient to counteract ABA’s inhibitory effects. Stirling’s greater sensitivity to ABA was further demonstrated by its significant germination inhibition at high ABA concentrations, consistent with previous findings that ABA sensitivity correlates with PHS resistance (Gao et al., 2023 ). Transcriptomic analyses confirmed upregulation of GA and ABA synthesis, metabolism, and signaling pathways, with NCED , AO, PP2C, PYR1, KAO1, GA3ox2, and DELLA emerging as key regulatory genes. Mutations in NCED were associated with variations in PHS tolerance, supporting its role in seed dormancy regulation (Lang et al., 2021 ). In addition to ABA and GA, other phytohormones were found to influence germination. Exogenous IAA exhibited a dual effect, promoting germination at low concentrations and inhibiting it at high concentrations, consistent with findings in wheat and tobacco (Ramaih et al., 2003 ; Li et al., 2016 ). Endogenous IAA levels decreased during germination and were higher in Stirling than Harrington, suggesting that IAA may interact with ABA to inhibit germination. The upregulation of YUCCA during germination indicates de novo IAA synthesis, with pan-genomic analyses identifying SNPs in YUCCA ( HORVU2Hr1G010060 ) and PVAs in ILR1 ( HORVU3Hr1G048560 ) and GH3 ( HORVU2Hr1G034670 ), which may contribute to germination differences. Cytokinins (CKs) also play a role in germination. Exogenous 6-BA inhibited barley germination, while endogenous trans-zeatin (tZ) levels increased in both varieties. The higher expression of LOG in Stirling suggests its involvement in CK biosynthesis, which may inhibit germination under certain conditions (Song et al., 2021 ). Jasmonates promote germination of dormant wheat kernels, and that methyl jasmonate inhibits the expression of the ABA biosynthesis gene ( TaNCED1 ), leading to a decrease in the ABA content prior to embryo germination (Jacobsen et al., 2013 ). Jasmonic acid (JA) showed opposing effects on germination, promoting it in Stirling while inhibiting it in Harrington. Transcriptomic data revealed high expression of JA biosynthesis genes, including LOX , AOS , and AOC , during germination, although JA content decreased over time, potentially due to metabolism. SA inhibited barley germination, with more pronounced effects in Harrington. Endogenous SA levels decreased during germination, paralleling ABA trends, suggesting SA may act as a negative regulator. Upregulation of SA biosynthesis genes ( ICS , PAL ) suggests active synthesis during germination while SA content decreased during germination in both barley varieties, and it may result from degradation or interactions with other hormones likely reduce its content. Under normal growth conditions, SA inhibits barley germination by suppressing GA-induced α-amylase gene expression (Xie et al., 2007 ). SA promotes seed germination of Arabidopsis thaliana under salt stress by reducing oxidative damage, and also promotes germination rate and germination potential of wheat and oat seeds under chromium stress (Lee et al., 2010 ; Wang et al., 2024a; Wang et al., 2024b). SA is synthesized through two pathways, ICS and PAL (Peng et al., 2021 ), and the genes encoding these synthetic enzymes ( ICS , EDS , EPS , and PAL ) were all up-regulated during seed germination, and ICS and PAL were high expression in two barley varieties. These findings offer insights into the physiological and molecular differences between fast- and slow-germinating barley varieties. Exogenous hormone treatments demonstrated that GA and ETH promote germination, while ABA, IAA, 6-BA, and SA inhibit it. JA inhibited germination in Harrington but promoted it in Stirling. Differences in endogenous ABA, GA1, IAA, and tZ levels may underlie these variations. Transcriptomic analyses identified 54 candidate genes, including NCED , AO1 , PP2C , PYR1 , KAO1 , GA3ox2 , DELLA , YUCCA , LOG , and others, as key regulators of germination. Pan-genomic analyses revealed 19 candidate genes with significant PAVs, which may contribute to genetic differentiation in germination traits. These findings provide a foundation for future studies on the molecular regulation of barley germination and the development of PHS-resistant barley varieties through molecular breeding. These candidate genes could be used to create novel functional alleles by fine-tuning gene expression and introduce the optimal allelic combination into elite cultivars using cutting-edge gene-editing technology. Abbreviations ABA Abscisic acid ACO ACC oxidase ACS ACC synthase ARF Auxin response factor AO Abscisic Aldehyde oxidase AOS Allene oxide synthase AUX Auxin BR Brassinolide CAR5 Developing grain (5 DAP) CAR15 Developing grain (15 DAP) CK Cytokinin CKX Cytokinin Oxidase CPS ent-Copalyl diphosphate synthase DELLA DELLA protein DET2 de-etiolated 2/steroid 5-alpha-reductase DMAPP Dimethylallyl pyrophosphate EDS Enhanced disease susceptibility EPS Enhanced pseudomonas susceptibility ETH Ethylene ETR2 Ethylene receptor 2 FPKM Fragments Per Kilobase of transcript per Million mapped reads GA Gibberellin GI Germination index GR Germination rate GR24 24-hour germination rate GR48 48-hour germination rate GH3 Acylamide synthetase GID1 Gibberellin Insensitive Dwarf 1 IAA Indole-3-acetic acid ICS Isochorismate synthase IPA Indole pyruvic acid JA Jasmonic acid LOX Lipoxygenase LOG Lonely Guy (cytokinin nucleoside 5′-monophosphoribose hydrolase) NCED 9-cis-Epoxycarotenoid dioxygenase NPR Nonexpresser of pathogenesis-related genes OPR 12-Oxophytodienoic acid reductase PAL Phenylalanine ammonia-lyase PAVs Presence/Absence variants PHS Pre-harvest sprouting PP2C group A type 2C protein phosphatase PYR1 Pyrabactin resistance 1 SA Salicylic acid S-AdoMets S-adenosylmethionine synthetase SNPs Single nucleotide polymorphisms tZ trans-Zeatin TIR/AFB Transport inhibitor response 1/Auxin signaling F-box proteins YUCCA Flavin-containing monooxygenase ZEP Zeaxanthin epoxidase Declarations Ethics approval and consent to participate The study did not involve human participants or animals, and thus, ethics approval and consent to participate were not applicable. All experiments were conducted using plant materials (barley seeds), and the research complied with relevant institutional and national guidelines for plant research. Consent for publication All authors have approved the final manuscript for publication and agree to its submission to the specified journal. Availability of data and materials All data supporting the findings of this study are available in the manuscript and supplementary material. Competing Interests The authors declare no competing interest. Funding Declaration This work is supported by the program provided by the Engineering Research Center of Ecology and Agricultural Use of Wetland, Ministry of Education KFT202302 and the funding from the National Natural Science Foundation of China (32372052). Authers Contributions Experimental design: Jun Wu, Bing Zhou, Bo Li, Yanhao Xu, Le Xu. Data generation, analysis and curation: Jun Wu, Bing Zhou, Peng Wang, Liya Luo, Ying Zhang. Project supervision: Le Xu. Manuscript draft: Jun Wu, Jiexin Zheng, Bo Li, Xu Le. Final manuscript: Yanhao Xu, Le Xu. Acknowledgements This work is supported by the program provided by the Engineering Research Center of Ecology and Agricultural Use of Wetland, Ministry of Education KFT202302 and the funding from the National Natural Science Foundation of China (32372052). References Betts, N.S., Dockter, C., Berkowitz, O., Collins, H., Hooi, M., Lu, Q. et al. 2020. Transcriptional and biochemical analyses of gibberellin expression and content in germinated barley grain. J Exp Bot. 71, 1870-1884. DOI: 10.1093/jxb/erz546. Bonnardeaux, Y., Li, C., Lance, R., Zhang, X., Sivasithamparam, K., Appels, R. 2008. Seed dormancy in barley: identifying superior genotypes through incorporating epistatic interactions. Aust J Agr Res. 59, 517-517. DOI:10.1071/ar07345. Gao, S., Zhou, H., Peng, X., Song, Y., He, X., Zhu, Y. 2023. Effects of physiological factors on dormancy of different seed coat-colored wheat varieties. 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Relationship of indoleacetic acid and tryptophan to dormancy and preharvest sprouting of wheat. Funct Plant Biol. 30(9), 939-945. DOI: 10.1071/FP03113. Shu, K., Liu, X., Xie, Q., He, Z. 2016. Two faces of one seed: hormonal regulation of dormancy and germination. Molecular Plant. 9, 34-45. DOI: 10.1016/j.molp.2015.08.010. Song, S., Liu, J., Yang, H., Zhang, W., Zhang, Q., Gao, J. 2021. Research progress in seed development, dormancy and germination regulated by cytokinin. Chinese Bulletin Bot . 56(02), 218-231. DOI: 10.11983/CBB20141. Spielmeyer, W., Ellis, M., Robertson, M., Ali, S., Lenton, J., Chandler, P. 2004. Isolation of gibberellin metabolic pathway genes from barley and comparative mapping in barley, wheat and rice. Theor Appl Genet. 109, 847-855. DOI: 10.1007/s00122-004-1689-6. Tai, L., Wang, H., Xu, X., Sun, W.H., Ju, L., Wenting, L. et al. 2021. Pre-harvest sprouting in cereals: genetic and biochemical mechanisms. J Exp Bot. 72, 2857-2876. DOI: 10.1093/jxb/erab024. Vetch, J., Stougaard, R., Martin, J., Giroux, M. 2019. Review: revealing the genetic mechanisms of pre-harvest sprouting in hexaploid wheat ( Triticum aestivum L.). Plant Sci. 281, 180-185. DOI: 10.1016/j.plantsci.2019.01.004. Xie, Z., Zhang, Z., Hanzlik, S., Cook, E., Shen, Q. 2007. Salicylic acid inhibits gibberellin-induced alpha-amylase expression and seed germination via a pathway involving an abscisic-acid-inducible WRKY gene. Plant Molecular Biol. 64, 293-303. DOI: 10.1007/s11103-007-9152-0. Xiong, M., Yu, J., Wang, J., Gao, Q., Huang, L., Chen, C. et al. 2022. Brassinosteroids regulate rice seed germination through the BZR1-RAmy3D transcriptional module. Plant Physiol. 189, 402-418. DOI: 10.1093/plphys/kiac043. Yan, B., Yang, Z., He, G., Jing, Y., Dong, H., Ju, L. et al. 2021. The blue light receptor CRY1 interacts with GID1 and DELLA proteins to repress gibberellin signaling and plant growth. Plant Commun. 2, 100245e. DOI: 10.1016/j.xplc.2021.100245. Yuan, J., Chen, D., Ren, Y., Zhang, X., Zhao, J. 2008. Characteristic and expression analysis of a metallothionein gene, OsMT2b , down-regulated by cytokinin suggests functions in root development and seed embryo germination of rice. Plant Physiol. 146, 1637-1650.DOI: 10.1104/pp.107.110304. Zeng, X., Guo, Y., Xu, Q., Mascher, M., Guo, G., Li, S. et al. 2018. Origin and evolution of qingke barley in Tibet. Nat Commun. 9, 5433. DOI: 10.1038/s41467-018-07920-5. Additional Declarations No competing interests reported. Supplementary Files SupplementaryTable18.xlsx Table S1 Germination rate and germination index of 236 barley varieties. Table S2 Phytohormone concentrations in exogenously treated barley. Table S3 RNA-seq data in the germinating embryo of Harrington and Stirling. Table S4 The primers used in the study. Table S5 Distribution of 24 h germination rate, 48 h germination rate, germination potential and germination index of 236 barley varieties. Table S6 Information about 11 varieties with germination index below 25. Table S7 Information of 12 varieties with germination index higher than 75. Table S8 Nineteen candidate genes with PAVs from the re-sequence data. FigureS1.pdf Fig. S1 Germination phenotypes and germination rates of Harrington and Stirling, different letters represent significant difference ( P <0.05). FigureS2.pdf Fig. S2 ETH and CK biosynthesis, signal transduction and metabolic pathways and genes expression. (A) Synthesis, metabolism and signaling gene expression of ETH during germination of Harrington and Stirling, S-AdoMets: S-adenosylmethionine synthetase, ACC: 1-aminocyclopropane-1-carboxylic acid, ACS: ACC synthase, ACO: ACC oxidase. (B) Expression of ETH synthesis, metabolism and signaling genes at 15 developmental stages in Morex. (C) Synthesis, metabolism and signaling gene expression of CK during germination of Harrington and Stirling, ADP, adenosine diphosphate; AMP: adenosine monophosphate, ATP: Adenosine triphosphate, CKX: CK oxidase/dehydrogenase, CYP735A: Cytochrome P450 monooxygenase, DMAPP: Dimethylallyl pyrophosphate, HK: histidine kinase, HP: histidine phosphate transfer protein, IPT: Isopentenyl transferase, LOG: CK nucleoside 5′-monophosphoribose hydrolase, SC: side chain, Type-B RRs: Type-B response regulator; Type-ARRs: Type-A response regulator. (D) Expression of CK synthesis, metabolism and signaling genes at 15 developmental stages in Morex. FigureS3.pdf Fig. S3 BR biosynthesis, signal transduction and metabolic pathways and genes expression. (A) DWF4: dwarf4/steroid 22α-hydroxylase, DET2: de-etiolated 2/steroid 5-alpha-reductase, CPD: constitutive photomorphogenesis and dwarfism/steroid 23-alpha-hydroxylase, ROT3: Cytochrome P450 90C1/Rotundifolia 3, CYP 90D1: Cytochrome P450 90D1, DWF1: Dwarf 1/Delta(24)-sterol reductase, BR6ox1: Brassinosteroid-6-oxidase 1, BSA1: phyB activation-tagged suppressor 1C, SMT1: Sterol methyltransferase, BRI1: Brassinosteroid insensitive 1, BKI1: BRI1 kinase inhibitor 1, BAK1: BRI1-associated receptor kinase 1, CDG1: Protein CONSTITUTIVE DIFFERENTIAL GROWTH 1, BSU1: Bri1 suppressor protein 1, BES1/BZR1: Brassinosteroid signaling positive regulator. (B) Expression of BR biosynthesis and signaling genes at 15 developmental stages in Morex. FigureS4.pdf Fig. S4 Real-time fluorescence quantification, different letters represent significant difference ( P <0.05). FigureS5.pdf Fig. S5 PAVs of GID1 , PP2C , BES1 , DELLA , TIR1/AFB , ARF , PAL6-5 and ILR1 in Morex, Barke, Stirling and Clipper. 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0.05).\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-6858206/v1/f43ad3350441ead0e0962998.png"},{"id":86792078,"identity":"351f02a0-091a-4798-a07a-66f6ac3d6e08","added_by":"auto","created_at":"2025-07-15 15:07:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":100480,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in endogenous ABA, GA1, IAA, \u003cem\u003et\u003c/em\u003eZ, JA, and SA content during Harrington versus Stirling germination, with different letters representing significant differences (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-6858206/v1/41a71c3fdc17447dcd8ccd08.png"},{"id":86792109,"identity":"7f87c94a-6baa-45c1-8888-a8abf074d62e","added_by":"auto","created_at":"2025-07-15 15:07:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":484431,"visible":true,"origin":"","legend":"\u003cp\u003eGA biosynthesis, signal transduction and metabolic pathways and genes expression. (A) Synthesis, metabolism and signaling gene expression of GA during germination of Harrington and Stirling, GGDP: geranylgeranyl diphosphate, \u003cem\u003eent\u003c/em\u003e-GDP: \u003cem\u003eent\u003c/em\u003e-copalyl diphosphate, CPS: \u003cem\u003eent\u003c/em\u003e-copalyl diphosphate synthase, KS: \u003cem\u003eent\u003c/em\u003e-kaurene synthase, KO: \u003cem\u003eent\u003c/em\u003e-kaurenoic oxidase, KAO: \u003cem\u003eent\u003c/em\u003e-kaurenoic acid oxidase, 13ox/20ox/2ox/3ox: GA13 oxidase /GA20 oxidase /GA2 oxidase /GA3 oxidase, GID: Gibberellin Insensitive Dwarf, GID1L: Gibberellin Insensitive Dwarf 1-like, DELLA: DELLA protein. (B) Expression of GA synthesis, metabolism and signaling genes at 15 developmental stages in Morex. EMB: 4-day embryos, ROO1: Roots from seedlings (10 cm shoot stage), LEA: Shoots from seedlings (10 cm shoot stage), INF2: Developing inflorescences (1-1.5 cm), NOD: Developing tillers, 3rd internode (42 DAP), CAR5: Developing grain (5 DAP), CAR15: Developing grain (15 DAP), ETI：Etiolated seedling, dark cond. (10 DAP), LEM: Inflorescences, lemma (42 DAP), LOD: Inflorescences, lodicule (42 DAP), DIN: Dissected inflorescences, palea (42 DAP), EPI: Epidermal strips (28 DAP), RAC: Inflorescences, rachis (35 DAP), ROO2: Roots (28 DAP), SEN: Senescing leaves (56 DAP). The same below.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-6858206/v1/296cbc616d898fdb6c1ca8c9.png"},{"id":86793162,"identity":"a609d184-0255-4bc5-917d-0a8cb0b22d94","added_by":"auto","created_at":"2025-07-15 15:15:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":618741,"visible":true,"origin":"","legend":"\u003cp\u003eABA biosynthesis, signal transduction and metabolic pathways and genes expression. (A) ABA synthesis, metabolism and signaling gene expression during germination of Harrington and Stirling. ZEP: Zeaxanthin epoxidase，NCED: 9-cis-epoxycarotenoid dioxygenase, XD: Xanthoxin dehydrogenase, AO: Abscisic Aldehyde oxidase, CYP707A: ABA 8'-hydroxylase, PYR1: Pyrabactin resistance 1, PP2C: group A type 2C protein phosphatase. (B) Expression of ABA synthesis, metabolism and signaling genes at 15 developmental stages in Morex.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-6858206/v1/7836c3983b6f9a94f6247031.png"},{"id":86793170,"identity":"f8e0281f-615e-4d74-89f4-012d06c66002","added_by":"auto","created_at":"2025-07-15 15:15:33","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":543759,"visible":true,"origin":"","legend":"\u003cp\u003eAUX biosynthesis, signal transduction and metabolic pathways and genes expression. (A) Synthesis, metabolism and signaling gene expression of IAA during germination of Harrington and Stirling, IND: Indole, TRP2/TSB1: Tryptophan synthase β-subunit, Trp: Tryptophan, TAM: Tryptamine, IAAld: Indole-3-acetaldehyde, IAA: Indole-3-acetic acid, AAOs: Aldehyde oxidase, TAAs: Tryptophan aminotransferase, YUCCA: Flavin-containing monooxygenase, VAS1: Pyridoxal phosphate dependent aminotransferase 1, IPA: Indole pyruvic acid, ILR1: Amide hydrolase, GH3: Acylamide synthetase. (B) Expression of IAA synthesis, metabolism and signaling genes at 15 developmental stages in Morex.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-6858206/v1/b41d0ec33208e91794223313.png"},{"id":86792082,"identity":"fb0fa49e-6214-46da-bf20-945a0e064f3b","added_by":"auto","created_at":"2025-07-15 15:07:31","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":607175,"visible":true,"origin":"","legend":"\u003cp\u003eJA and SA biosynthesis, signal transduction and metabolic pathways and genes expression. (A) Synthesis, metabolism and signaling gene expression of JA during germination of Harrington and Stirling: LOX: Lipoxygenase, AOS: Allene oxide synthase, AOC: Allene oxide cyclase, OPR: 12-oxo-phytodienoic acid reductase, OPC-8: (+/-)-12-Oxodihydrophytodienoic acid，JAR1: Jasmonamide synthase，COI1: COR-insensitive 1, JAZ: Jasmonate-zim-domain protein, MYC2:Transcription factor MYC2. (B) Expression of JA synthesis, metabolism and signaling genes at 15 developmental stages in Morex. (C) Synthesis, metabolism and signaling gene expression of SA during germination of Harrington and Stirling, IC: Isochorismate, ICS: Isochorismate synthase, IC-9-Glu：Isomeric acid -9-glutamic acid, EDS: Enhanced disease susceptibility, EPS: Enhanced pseudomonas susceptibility, Phe: Phenylalanine, PAL: Phenylalanine ammonia-lyase, NPR: Nonexpresser of pathogenesis-related genes, TGA: Transcription factor. (D) Expression of SA synthesis, metabolism and signaling genes at 15 developmental stages in Morex.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-6858206/v1/94ffb8782b456c899f995ddd.png"},{"id":86792096,"identity":"75293900-1801-47bd-aac1-238f30405418","added_by":"auto","created_at":"2025-07-15 15:07:32","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":536931,"visible":true,"origin":"","legend":"\u003cp\u003ePAVs of \u003cem\u003eKAO1\u003c/em\u003e, \u003cem\u003eYUCCA\u003c/em\u003eand \u003cem\u003eNCED\u003c/em\u003e in Morex, Barke, Stirling and Clipper.\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-6858206/v1/f5b2c5d47ac9cc07128792e4.png"},{"id":88889042,"identity":"636b65ed-f942-45d1-a091-1b66dfeec4e2","added_by":"auto","created_at":"2025-08-12 12:31:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3987719,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6858206/v1/d58ec915-557a-4497-b358-241aad85c229.pdf"},{"id":86793163,"identity":"0c528252-f053-423d-b834-921f7b6842d2","added_by":"auto","created_at":"2025-07-15 15:15:31","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":66833,"visible":true,"origin":"","legend":"\u003cp\u003eTable S1 Germination rate and germination index of 236 barley varieties.\u003c/p\u003e\n\u003cp\u003eTable S2 Phytohormone concentrations in exogenously treated barley.\u003c/p\u003e\n\u003cp\u003eTable S3 RNA-seq data in the germinating embryo of Harrington and Stirling.\u003c/p\u003e\n\u003cp\u003eTable S4 The primers used in the study.\u003c/p\u003e\n\u003cp\u003eTable S5 Distribution of 24 h germination rate, 48 h germination rate, germination potential and germination index of 236 barley varieties.\u003c/p\u003e\n\u003cp\u003eTable S6 Information about 11 varieties with germination index below 25.\u003c/p\u003e\n\u003cp\u003eTable S7 Information of 12 varieties with germination index higher than 75.\u003c/p\u003e\n\u003cp\u003eTable S8 Nineteen candidate genes with PAVs from the re-sequence data.\u003c/p\u003e","description":"","filename":"SupplementaryTable18.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6858206/v1/f0364f07f9345d0b6b9268ca.xlsx"},{"id":86793169,"identity":"8754c390-1676-467f-a7b9-a3dbd0af1150","added_by":"auto","created_at":"2025-07-15 15:15:33","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":108171,"visible":true,"origin":"","legend":"\u003cp\u003eFig. S1 Germination phenotypes and germination rates of Harrington and Stirling, different letters represent significant difference (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"FigureS1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6858206/v1/f56d39c1b3be9c0b4309b774.pdf"},{"id":86792081,"identity":"ee78ad6b-9965-46e9-a380-ee14f585f6cb","added_by":"auto","created_at":"2025-07-15 15:07:31","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":328644,"visible":true,"origin":"","legend":"\u003cp\u003eFig. S2 ETH and CK biosynthesis, signal transduction and metabolic pathways and genes expression. (A) Synthesis, metabolism and signaling gene expression of ETH during germination of Harrington and Stirling, S-AdoMets: S-adenosylmethionine synthetase, ACC: 1-aminocyclopropane-1-carboxylic acid, ACS: ACC synthase, ACO: ACC oxidase. (B) Expression of ETH synthesis, metabolism and signaling genes at 15 developmental stages in Morex. (C) Synthesis, metabolism and signaling gene expression of CK during germination of Harrington and Stirling, ADP, adenosine diphosphate; AMP: adenosine monophosphate, ATP: Adenosine triphosphate, CKX: CK oxidase/dehydrogenase, CYP735A: Cytochrome P450 monooxygenase, DMAPP: Dimethylallyl pyrophosphate, HK: histidine kinase, HP: histidine phosphate transfer protein, IPT: Isopentenyl transferase, LOG: CK nucleoside 5′-monophosphoribose hydrolase, SC: side chain, Type-B RRs: Type-B response regulator; Type-ARRs: Type-A response regulator. (D) Expression of CK synthesis, metabolism and signaling genes at 15 developmental stages in Morex.\u003c/p\u003e","description":"","filename":"FigureS2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6858206/v1/f228eed7b212a0e7e81398b0.pdf"},{"id":86792084,"identity":"92b4bdfc-3893-4fa0-b076-52d0bf7b2b46","added_by":"auto","created_at":"2025-07-15 15:07:31","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":488705,"visible":true,"origin":"","legend":"\u003cp\u003eFig. S3 BR biosynthesis, signal transduction and metabolic pathways and genes expression. (A) DWF4: dwarf4/steroid 22α-hydroxylase, DET2: de-etiolated 2/steroid 5-alpha-reductase, CPD: constitutive photomorphogenesis and dwarfism/steroid 23-alpha-hydroxylase, ROT3: Cytochrome P450 90C1/Rotundifolia 3, CYP 90D1: Cytochrome P450 90D1, DWF1: Dwarf 1/Delta(24)-sterol reductase, BR6ox1: Brassinosteroid-6-oxidase 1, BSA1: phyB activation-tagged suppressor 1C, SMT1: Sterol methyltransferase, BRI1: Brassinosteroid insensitive 1, BKI1: BRI1 kinase inhibitor 1, BAK1: BRI1-associated receptor kinase 1, CDG1: Protein CONSTITUTIVE DIFFERENTIAL GROWTH 1, BSU1: Bri1 suppressor protein 1, BES1/BZR1: Brassinosteroid signaling positive regulator. (B) Expression of BR biosynthesis and signaling genes at 15 developmental stages in Morex.\u003c/p\u003e","description":"","filename":"FigureS3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6858206/v1/7445002e9f6cc7c4a5af966f.pdf"},{"id":86792085,"identity":"abea04a6-b5ac-4823-a3d3-33ecf7ce7346","added_by":"auto","created_at":"2025-07-15 15:07:31","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":180497,"visible":true,"origin":"","legend":"\u003cp\u003eFig. S4 Real-time fluorescence quantification, different letters represent significant difference (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"FigureS4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6858206/v1/ff275ac6fe97673e8edbe428.pdf"},{"id":86792086,"identity":"e6b313e4-0975-4403-bb5e-e9bd86db108b","added_by":"auto","created_at":"2025-07-15 15:07:31","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":536256,"visible":true,"origin":"","legend":"\u003cp\u003eFig. S5 PAVs of \u003cem\u003eGID1\u003c/em\u003e, \u003cem\u003ePP2C\u003c/em\u003e, \u003cem\u003eBES1\u003c/em\u003e, \u003cem\u003eDELLA\u003c/em\u003e, \u003cem\u003eTIR1/AFB\u003c/em\u003e, \u003cem\u003eARF\u003c/em\u003e, \u003cem\u003ePAL6-5\u003c/em\u003e and \u003cem\u003eILR1\u003c/em\u003e in Morex, Barke, Stirling and Clipper.\u003c/p\u003e","description":"","filename":"FigureS5.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6858206/v1/da29025c704bb40fb8c21c44.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Molecular mechanism of phytohormones regulating dormancy and germination of barley seeds","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eDormancy, defined as the incapacity of an intact viable seed to complete germination under favourable conditions. Weak seed dormancy in barley leads to pre-harvest sprouting (PHS), but strong dormancy results in poor malting quality, which causes significant economic loss worldwide cereal-growing regions (Tai et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). PHS occurs in almost every cereal-growing region globally and is responsible for up to \u003cspan\u003e$\u003c/span\u003e1\u0026nbsp;billion in annual losses (Miao et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Lang et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Vetch et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSeed dormancy and germination are crucial traits in cereals, as seeds lacking dormancy are highly susceptible to PHS (Bonnardeaux et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Domestication and modern breeding practices aim to promote rapid and uniform plant establishment but often reduce grain dormancy, making crops more susceptible to PHS (Newberry et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Phytohormones such as gibberellin (GA), ethylene (ETH), brassinolide (BR), and cytokinin (CK) promote seed germination and positively regulate PHS, while abscisic acid (ABA), auxin, salicylic acid (SA), and jasmonic acid (JA) negatively regulate PHS by inhibiting germination (Yan et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe application of GA enhances seed germination and alleviates dormancy (Nonogaki, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). For example, the transcriptional abundance of genes such as \u003cem\u003eKO1\u003c/em\u003e, \u003cem\u003eKAO1\u003c/em\u003e, \u003cem\u003eGA20ox\u003c/em\u003e, \u003cem\u003eGA3ox\u003c/em\u003e, and \u003cem\u003eGID1b\u003c/em\u003e increases during GA synthesis in barley germination (Betts et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). ETH similarly promotes germination and dormancy release, as exogenous ETH or ethephon can break both primary and secondary seed dormancy (Li et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Other hormones, such as BR and CK, also play critical roles in regulating germination through signaling pathways (Xiong et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Yuan et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). ABA levels decrease during seed imbibition, facilitating germination in both barley and \u003cem\u003eArabidopsis\u003c/em\u003e (Jacobsen et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Millar et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Auxin enhances ABA-induced dormancy by interacting with ARF (auxin response factor) transcription factors to maintain \u003cem\u003eABI3\u003c/em\u003e and \u003cem\u003eABI5\u003c/em\u003e expression during seed imbibition (Liu et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Similarly, SA and JA regulate germination under stress conditions (Li et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Ju et al., 2019).\u003c/p\u003e\u003cp\u003eBarley (\u003cem\u003eHordeum vulgare\u003c/em\u003e L.) is an ancient crop that ranks fourth globally in terms of cultivated area and total production (Zeng et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eUnderstanding the molecular mechanisms underlying phytohormone regulation of barley seed dormancy and germination is critical to fine-tune PHS and rapid germination. We need to create an ideotype with an adequate dormancy level to prevent PHS but capable of rapid and efficient germination under the right conditions shortly after harvest; this will provide high malting quality. We propose that fine-tuning the genes involved in grain dormancy, seed germination, and malting quality will likely achieve the outcome. This study used two barley varieties, the PHS-sensitive Harrington and the PHS-resistant Stirling, to investigate the transcriptional and physiological changes during germination. By analyzing phytohormone regulation and gene expression during germination, the findings of this study provide important insights into the molecular basis of PHS regulation and offer valuable information for breeding PHS-resistant barley varieties suitable for high rainfall and humidity conditions.\u003c/p\u003e"},{"header":"2. MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec2\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Characterization of germination in different genotypes of barley\u003c/h2\u003e\u003cp\u003eDifferent barley varieties were kindly provided by Prof. Meixue Zhou from University of Tasmania. Uniform barley seeds were sterilized with 75% ethanol for 1 minute, followed by 2% sodium hypochlorite for 15 minutes, washed with sterile water, and placed in 9 cm petri dishes with two layers of filter paper. A total of 100 seeds were placed per dish, and 4 mL of distilled water were added to each petri dish, and samples were incubated in the dark at 20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026deg;C. Germination rate (GR) was measured at 24 h, 48 h and 72 h, and germination index (GI) were measured at 72 h.\u003c/p\u003e\u003cp\u003eGermination index (GI)=\u0026sum;(Gt/Dt), Gt is the number of germinated seeds day by day, and Dt is the number of days to germination for the corresponding DG, counted up to day 3.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Exogenous hormone treatment of Harrington and Stirling\u003c/h2\u003e\u003cp\u003eFour milliliters of hormone solution at specified concentrations (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e) were added to each petri dish, with distilled water serving as a control. Dishes were incubated in the dark at 20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026deg;C. The number of germinated seeds (the appearance of white radicle) was recorded at 24 h, 36 h, 48 h, 60 h, and 72 h.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Measurement of endogenous phytohormones\u003c/h2\u003e\u003cp\u003ePhytohormone quantification was conducted using an ESI-HPLC-MS/MS (Electrospray ionization-high-performance liquid chromatography-mass spectrometry/mass spectrometry) system equipped with a QTRAP\u0026trade; 6500 liquid-mass spectrometer (AB Sciex). Full-grain Harrington and Stirling seeds were sampled 24 h and 48 h after incubation. Only seed embryos were collected, immediately frozen on dry ice, and stored in an ultra-low-temperature freezer at \u0026minus;\u0026thinsp;80\u0026deg;C. Each sample contained 100\u0026ndash;300 embryos, for hormone content analysis. Standard solutions of IAA, ABA, JA, SA, GA1, and \u003cem\u003et\u003c/em\u003eZ were prepared in methanol at concentrations ranging from 0.1 ng/mL to 200 ng/mL. The linear standard curves were plotted.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Expression analysis of phytohormone-related genes\u003c/h2\u003e\u003cp\u003eTranscriptomic differences in Harrington and Stirling barley were analyzed at 24 h and 48 h of germination using publicly available data (Zhang et al., 2016; accession number GSE66024). Gene expression was quantified as Fragments Per Kilobase of transcript per Million mapped reads (FPKM). Data on Morex barley gene expression during 15 growth stages were obtained to identify germination-specific genes (Mayer et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Candidate genes involved in GA, ABA, AUX, CTK, ETH, BR, JA, and SA synthesis, metabolism, and signaling were identified from previous reports (Spielmeyer et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Shu et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Nguyen et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Sequences of phytohormone-related genes reported in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, wheat, rice, and barley were combined with barley genome annotations and GenBank data. A total of 260 phytohormone-related genes were identified through BLAST-based nucleotide and protein homology searches (Mascher et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Detailed information on these genes is provided in Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Pan-genome sequence analysis\u003c/h2\u003e\u003cp\u003eCandidate genes identified in the transcriptome analyses were compared using pan-genomic data from four barley varieties (Barke, Morex, Stirling, and Clipper) (Jayakodi et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Hu et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Barke and Morex germinated quickly while, the germination index of Stirling and Clipper was below 25, which is highly PHS resistant.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.6 RNA extraction and real-time fluorescence quantification\u003c/h2\u003e\u003cp\u003eRNA was extracted using the TRIzol method. RNA quality was assessed using a NanoDrop 2000 UV spectrophotometer and 1% agarose gel electrophoresis. High-quality RNA was stored at \u0026minus;\u0026thinsp;80\u0026deg;C. cDNA synthesis was performed using the HiScript\u0026reg; II 1st Strand cDNA Synthesis Kit (+\u0026thinsp;gDNA wiper). Quantitative RT-PCR was conducted on a QuantStudio\u0026trade; 7 Flex Real-Time PCR System (Applied Biosystems, USA) with a ChamQ SYBR qPCR Master Mix (Vazyme, China). Candidate gene primers were designed using Primer Premier 5 and validated with the NCBI database (Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e"},{"header":"3. RESULTS","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Germination characteristics of 236 barley germplasm resources\u003c/h2\u003e\u003cp\u003eIn this study, the germination characteristics of 236 barley germplasm resources were assessed using germination rate (24 h-GR24, 48 h-GR48), and germination index (GI). The germination rate and germination index data for the 236 barley varieties are presented in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003eGR24 ranged from 1.00\u0026ndash;96.67%, GR48 from 7.67\u0026ndash;97.00%, and GI from 5.22 to 97.17. The coefficients of variation for these indices were 82.80%, 40.61%, and 41.24%, respectively, indicating extensive germination traits variation and genetic diversity of this population (Table \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e). Among the 236 varieties, in 2022 and 2023, the germination index of MSS87, 261, Stirling, Clipper, C2-05-337-2, Irene, 262, Linus, Troon, 260, and USA 8330\u0026thinsp;\u0026minus;\u0026thinsp;1679 were below 25 (Table \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e). Conversely, 12 varieties showed fast germination, such as SHEBA, ETHIOPIA 602, Harrington, Morex, and Barke (Table S7). PHS sensitive Harrington, and PHS tolerant-Stirling were selected for subsequent analyses due to their significant differences in germination traits. At 24 h, Harrington exhibited a germination rate of 62.33%, significantly higher than Stirling (around 1%) (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Effects of exogenous hormone treatment on germination of Harrington and Stirling\u003c/h2\u003e\u003cp\u003eExogenous GA3 significantly increased the germination of Harrington and Stirling with increasing concentrations and ABA inhibited germination (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). ETH treatment increased Stirling\u0026rsquo;s germination significantly while Harrington did not response to ETH (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). IAA and EBR showed dual effects: promoting germination at low concentrations and inhibiting it at higher concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eD and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). JA had no significant impact on Harrington but increased Stirling\u0026rsquo;s germination (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Both 6-BA and SA inhibited the germination of Harrington and Stirling in a dose dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eG and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eH).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Endogenous Hormone Content during Germination\u003c/h2\u003e\u003cp\u003eEndogenous levels of ABA, GA1, IAA, tZ (trans-zeatin), JA, and SA were measured in Harrington and Stirling embryos at 24 h and 48 h using ESI-HPLC-MS/MS (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e). GA1 levels increased in both varieties during germination, with significantly higher levels in Stirling than Harrington at both time points (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). ABA level decreased significantly during germination and it was lower in Harrington than Stirling at 24 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eIAA levels also decreased during germination and were consistently higher in Stirling than in Harrington, suggesting a potential role in the observed germination differences (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The tZ content increased in both varieties from 24 h to 48 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). JA and SA levels decreased during germination in both varieties (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eE and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eF).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Identification of Key Differential Expressed Genes (DEGs) in Phytohormone Pathways\u003c/h2\u003e\u003cp\u003eUsing transcriptomic data from Harrington and Stirling at 24 h and 48 h of germination, we chose differential Expressed Genes (DEGs) according to three criterial: firstly, these genes should have roles in phytohormone biosynthesis, metabolism or signaling; secondly, different expression pattern in Harrington and Stirling (fold change\u0026thinsp;\u0026ge;\u0026thinsp;2); thirdly, these genes were expressed specific at germination stage (4-day embryos, EMB) based on the germination data from 15 developmental stages of Morex (Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn the GA pathway, genes such as \u003cem\u003eKAO1\u003c/em\u003e (\u003cem\u003eent\u003c/em\u003e-kaurenoic acid oxidase), \u003cem\u003eGID1\u003c/em\u003e (\u003cem\u003eHORVU1Hr1G060810\u003c/em\u003e), and \u003cem\u003eDELLA\u003c/em\u003e (\u003cem\u003eHORVU4Hr1G006930\u003c/em\u003e) exhibited higher expression in Stirling than Harrington during germination (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). In the Morex 4-day embryos (EMB), elevated expression levels of \u003cem\u003eKAO1\u003c/em\u003e (\u003cem\u003eHORVU7Hr1G003090\u003c/em\u003e), \u003cem\u003eGA13ox2\u003c/em\u003e (\u003cem\u003eHORVU4Hr1G050930\u003c/em\u003e), and \u003cem\u003eGA3ox2\u003c/em\u003e (\u003cem\u003eHORVU3Hr1G022840\u003c/em\u003e) were detected. This upregulation may correlate with the observed increase in GA1 content in germinating Harrington and Stirling (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eSimilar trends were observed for ABA-related genes such as \u003cem\u003eNCED\u003c/em\u003e (9-cis-epoxycarotenoid dioxygenase, \u003cem\u003eHORVU5Hr1G000320\u003c/em\u003e), which were upregulated in Stirling (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Abscisic aldehyde oxidase (AO) is the last key enzyme in the ABA synthesis pathway, and expression of \u003cem\u003eAO\u003c/em\u003e (\u003cem\u003eHORVU5Hr1G103890\u003c/em\u003e, \u003cem\u003eHORVU7Hr1G114000\u003c/em\u003e, \u003cem\u003eHORVU7Hr1G114020\u003c/em\u003e) is high in EMB of Morex (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). \u003cem\u003ePP2C\u003c/em\u003e (\u003cem\u003eHORVU5Hr1G097060\u003c/em\u003e, \u003cem\u003eHORVU3Hr1G085930\u003c/em\u003e), and \u003cem\u003ePYR1\u003c/em\u003e (\u003cem\u003eHORVU4Hr1G055220\u003c/em\u003e, \u003cem\u003eHORVU1Hr1G050110\u003c/em\u003e) in the ABA signaling pathway had higher expression in EMB (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The transcript expression of \u003cem\u003ePYR1\u003c/em\u003e and \u003cem\u003ePP2C\u003c/em\u003e in the Stirling were significantly higher than in Harrington (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003eThe YUCCA family encodes flavin monooxygenase, which is the rate-limiting enzyme in auxin synthesis. The expression of most \u003cem\u003eYUCCA\u003c/em\u003e transcripts were up-regulated from 24 h to 48 h during germination, and \u003cem\u003eYUCCA\u003c/em\u003e expression were significantly higher in Stirling than in Harrington (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). \u003cem\u003eYUCCA\u003c/em\u003e (\u003cem\u003eHORVU2Hr1G010060\u003c/em\u003e, \u003cem\u003eHORVU7Hr1G027520\u003c/em\u003e) were highly expressed during the EMB period in Morex (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). \u003cem\u003eARF\u003c/em\u003e (auxin response factor, \u003cem\u003eHORVU6Hr1G026730\u003c/em\u003e, \u003cem\u003eHORVU7Hr1G108360\u003c/em\u003e, \u003cem\u003eHORVU7Hr1G051930\u003c/em\u003e), \u003cem\u003eTIR/AFB\u003c/em\u003e (\u003cem\u003eHORVU1Hr1G021550\u003c/em\u003e, \u003cem\u003eHORVU2Hr1G070800\u003c/em\u003e) and \u003cem\u003eAUX/IAA\u003c/em\u003e (\u003cem\u003eHORVU1Hr1G086070\u003c/em\u003e, \u003cem\u003eHORVU3Hr1G031460\u003c/em\u003e, \u003cem\u003eHORVU1Hr1G025670\u003c/em\u003e) were highly expressed during EMB (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). These eight genes were significantly different between Harrington and Stirling (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The expression of \u003cem\u003eGH3\u003c/em\u003e (\u003cem\u003eHORVU2Hr1G03467\u003c/em\u003e) and \u003cem\u003eILR1-like\u003c/em\u003e (\u003cem\u003eHORVU3Hr1G048560, HORVU3Hr1G066450\u003c/em\u003e) were high during EMB in Morex (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Auxin-related genes showed higher expression in Stirling, indicating their involvement in regulating germination (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003eThe \u003cem\u003eS-AdoMets\u003c/em\u003e (\u003cem\u003eHORVU6Hr1G063540\u003c/em\u003e, \u003cem\u003eHORVU6Hr1G063490\u003c/em\u003e), \u003cem\u003eACS\u003c/em\u003e (\u003cem\u003eHORVU3Hr1G019430\u003c/em\u003e, \u003cem\u003eHORVU3Hr1G019410\u003c/em\u003e), \u003cem\u003eACO\u003c/em\u003e (\u003cem\u003eHORVU5Hr1G067530\u003c/em\u003e), and \u003cem\u003eETR2\u003c/em\u003e (\u003cem\u003eHORVU5Hr1G115160\u003c/em\u003e) accumulated in EMB (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Also in Harrington and Stirling, transcript expression of \u003cem\u003eACS\u003c/em\u003e were up-regulated from 24 h to 48 h of germination, and the six \u003cem\u003eACO\u003c/em\u003e were up-regulated from 24 h to 48 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn BR biosynthesis, signaling and metabolic pathways, the expression of \u003cem\u003eDET2\u003c/em\u003e (\u003cem\u003eHORVU4Hr1G060850\u003c/em\u003e) and \u003cem\u003eBR6ox1\u003c/em\u003e (\u003cem\u003eHORVU2Hr1G002250\u003c/em\u003e) were significantly higher in Stirling during germination than in Harrington (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eA). \u003cem\u003eDWF1\u003c/em\u003e (\u003cem\u003eHORVU7Hr1G120030\u003c/em\u003e), \u003cem\u003eBAK1\u003c/em\u003e (\u003cem\u003eHORVU6Hr1G049080\u003c/em\u003e), and \u003cem\u003eBES1/BZR1\u003c/em\u003e (\u003cem\u003eHORVU0Hr1G040070\u003c/em\u003e, \u003cem\u003eHORVU7Hr1G088980\u003c/em\u003e) were significantly higher in Harrington than in Stirling (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eA). Higher expression of \u003cem\u003eDET2\u003c/em\u003e, \u003cem\u003eDWF1\u003c/em\u003e, \u003cem\u003eBR6ox1\u003c/em\u003e, \u003cem\u003eBSU1\u003c/em\u003e, \u003cem\u003eBAK1\u003c/em\u003e and \u003cem\u003eBES1/BZR1\u003c/em\u003e were found during EMB in Morex (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eThe transcript expression of \u003cem\u003eLOG\u003c/em\u003e (Lonely Guy, \u003cem\u003eHORVU2Hr1G089620\u003c/em\u003e), \u003cem\u003eCKX\u003c/em\u003e (Cytokinin Oxidase, \u003cem\u003eHORVU3Hr1G019850\u003c/em\u003e) and \u003cem\u003eARR\u003c/em\u003e (Arabidopsis Response Regulator, \u003cem\u003eHORVU6Hr1G028680\u003c/em\u003e, \u003cem\u003eHORVU3Hr1G114970\u003c/em\u003e) in CK biosynthesis, signaling and metabolism pathway was significantly higher in Stirling than in Harrington (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eC). Among them, \u003cem\u003eHORVU2Hr1G089620\u003c/em\u003e was down-regulated from 24 h to 48 h (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eC). The transcript expression of these four genes was higher in expression during the EMB period (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003eJA biosynthesis, signaling, and metabolism-related \u003cem\u003eLOX\u003c/em\u003e (Lipoxygenase, \u003cem\u003eHORVU5Hr1G093770\u003c/em\u003e, \u003cem\u003eHORVU6Hr1G000510\u003c/em\u003e, \u003cem\u003eHORVU5Hr1G093700\u003c/em\u003e, and \u003cem\u003eHORVU4Hr1G005920\u003c/em\u003e), \u003cem\u003eAOS\u003c/em\u003e (Allene Oxide Synthase, \u003cem\u003eHORVU4Hr1G066270\u003c/em\u003e, \u003cem\u003eHORVU6Hr1G039440\u003c/em\u003e), \u003cem\u003eAOC\u003c/em\u003e (Allene Oxide Cyclase, \u003cem\u003eHORVU6Hr1G081000\u003c/em\u003e) and \u003cem\u003eOPR\u003c/em\u003e (12-Oxophytodienoic Acid Reductase, \u003cem\u003eHORVU2Hr1G004230\u003c/em\u003e, \u003cem\u003eHORVU7Hr1G036070\u003c/em\u003e) showed lower expression in Harrington than Stirling (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Among them, \u003cem\u003eLOX\u003c/em\u003e and \u003cem\u003eAOS\u003c/em\u003e had higher transcript expression during EMB (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eSA biosynthesis includes the isochorismate synthase (\u003cem\u003eICS\u003c/em\u003e) pathway and the phenylalanine ammonia-lyase (\u003cem\u003ePAL\u003c/em\u003e) pathway. The transcript expression of \u003cem\u003eICS\u003c/em\u003e and \u003cem\u003ePAL\u003c/em\u003e in both Harrington and Stirling was up-regulated during germination at 24 h and 48 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). \u003cem\u003eICS\u003c/em\u003e (\u003cem\u003eHORVU5Hr1G057050\u003c/em\u003e), \u003cem\u003eEPS1\u003c/em\u003e (\u003cem\u003eHORVU1Hr1G072050\u003c/em\u003e), \u003cem\u003ePAL2\u003c/em\u003e (\u003cem\u003eHORVU0Hr1G016330\u003c/em\u003e), and \u003cem\u003ePAL6-5\u003c/em\u003e (\u003cem\u003eHORVU2Hr1G089440\u003c/em\u003e) were highly expressed in barley during the EMB period (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). The high transcript levels of genes within the ICS and PAL pathways indicate active SA synthesis during the germination, implying a potential regulatory role in the germination process.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Validation of Differential Gene Expression\u003c/h2\u003e\u003cp\u003eEight differentially expressed genes (\u003cem\u003eHORVU4Hr1G006930\u003c/em\u003e, \u003cem\u003eHORV5Hr1G103890\u003c/em\u003e, \u003cem\u003eHORV7Hr1G027520\u003c/em\u003e, \u003cem\u003eHORV6Hr1G03450\u003c/em\u003e, \u003cem\u003eHORV6Hr1G06390\u003c/em\u003e, \u003cem\u003eHORV4Hr1G060850\u003c/em\u003e, \u003cem\u003eHORV4Hr1G066270\u003c/em\u003e and \u003cem\u003eHORV0Hr1G016330\u003c/em\u003e) involved in phytohormone synthesis and metabolism were selected for RT-qPCR validation. Results were consistent with transcriptomic data, confirming the reliability of RNA-seq findings. Genes related to AUX, CK, and ABA were identified as key regulators of germination in barley (Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.6 Pan-genome reveals candidate gene variances\u003c/h2\u003e\u003cp\u003eAll of the above genes were induced during germination and they show distinct expression pattern in Harrington and Stirling germination. Accordingly, we conducted a comparative analysis of the 54 genes against the published barley pan-genome to ascertain the presence of any variations within the coding DNA sequence (CDS) region and to explore the potential correlation between these variations and the differential sprouting behaviors observed.\u003c/p\u003e\u003cp\u003eHu et al \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e. found that significant PAVs (Presence/Absence variants) occurred in genes involved in photoperiod, flowering time through comparative genomics studies of barley in Europe, Australia and North America. Whether barley varieties with different germination rates differ significantly in expression due to PAVs during the long evolution of barley? We compared the presence of PAVs in 54 candidate genes selected from the transcriptome results by four barley varieties in the pan-genomic study: Barke (germination index 88, fast germination), Morex (germination index 85, fast germination), Stirling (germination index 21, slow germination), and Clipper (germination index 23, slow germination). We found that there were only 2 genes with SNPs (single nucleotide polymorphisms) in the CDS region as \u003cem\u003eKAO1\u003c/em\u003e (\u003cem\u003eHORVU7Hr1G003090\u003c/em\u003e) and \u003cem\u003eYUCCA\u003c/em\u003e (\u003cem\u003eHORVU2Hr1G010060\u003c/em\u003e).\u003c/p\u003e\u003cp\u003eThe \u003cem\u003eNCED\u003c/em\u003e (\u003cem\u003eHORVU5Hr1G000320\u003c/em\u003e) has multiple base substitutions starting at position 316 bp, and it cause the amino acid sequence change, which may lead to changes in its expression in different barley varieties (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e7\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003eThere were insertions or deletions of large segments in the CDS region of 17 genes, as shown in Table S8. We found a 3bp insertion and deletion of \u003cem\u003eGID1\u003c/em\u003e, \u003cem\u003ePP2C\u003c/em\u003e and \u003cem\u003eBES1\u003c/em\u003e in Stirling and Clipper (Fig. \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003eA, B and C). \u003cem\u003eDELLA\u003c/em\u003e, \u003cem\u003eTIR1/AFB\u003c/em\u003e, \u003cem\u003eARF\u003c/em\u003e, \u003cem\u003ePAL6-5\u003c/em\u003e, and \u003cem\u003eILR1\u003c/em\u003e all produced large segment deletions (Fig. \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003eD, E, F, G, and H). Our results suggest that the 19 candidate genes that are highly or specifically expressed during seed germination have different PAVs, which may lead to genetic differentiation of germination characteristics. These variations likely contribute to differences in germination speed and hormone sensitivity among barley varieties (Table S8; Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e7\u003c/span\u003e)\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eBarley is a widely cultivated crop with high nutritional value, but PHS significantly contributes to yield losses. Recent studies suggest that phytohormone engineering could mitigate PHS, as genes regulating seed dormancy and germination are significantly enriched in phytohormone metabolic pathways (Nonogaki, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Understanding the mechanisms by which phytohormones regulate seed dormancy and germination is essential for developing effective strategies to address PHS. This study aimed to elucidate the molecular mechanisms underlying phytohormone metabolism and signaling during barley seed germination under controlled conditions. To achieve this, we evaluated the effects of exogenous phytohormones on barley germination, quantified endogenous phytohormones using ESI-HPLC-MS/MS, and identified key genes in phytohormone pathways through transcriptomic and pan-genomic analyses.\u003c/p\u003e\u003cp\u003eIn this study, exogenous GA3 significantly promoted barley germination, while exogenous ABA inhibited it. Endogenous hormone assays revealed that ABA and GA1 levels increased from 24 h to 48 h during germination in both Harrington and Stirling. Stirling had higher ABA and GA1 levels than Harrington, indicating that its endogenous GA1 levels were insufficient to counteract ABA\u0026rsquo;s inhibitory effects. Stirling\u0026rsquo;s greater sensitivity to ABA was further demonstrated by its significant germination inhibition at high ABA concentrations, consistent with previous findings that ABA sensitivity correlates with PHS resistance (Gao et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Transcriptomic analyses confirmed upregulation of GA and ABA synthesis, metabolism, and signaling pathways, with \u003cem\u003eNCED\u003c/em\u003e, AO, PP2C, PYR1, KAO1, GA3ox2, and DELLA emerging as key regulatory genes. Mutations in NCED were associated with variations in PHS tolerance, supporting its role in seed dormancy regulation (Lang et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn addition to ABA and GA, other phytohormones were found to influence germination. Exogenous IAA exhibited a dual effect, promoting germination at low concentrations and inhibiting it at high concentrations, consistent with findings in wheat and tobacco (Ramaih et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Endogenous IAA levels decreased during germination and were higher in Stirling than Harrington, suggesting that IAA may interact with ABA to inhibit germination. The upregulation of YUCCA during germination indicates de novo IAA synthesis, with pan-genomic analyses identifying SNPs in YUCCA (\u003cem\u003eHORVU2Hr1G010060\u003c/em\u003e) and PVAs in \u003cem\u003eILR1\u003c/em\u003e (\u003cem\u003eHORVU3Hr1G048560\u003c/em\u003e) and \u003cem\u003eGH3\u003c/em\u003e(\u003cem\u003eHORVU2Hr1G034670\u003c/em\u003e), which may contribute to germination differences.\u003c/p\u003e\u003cp\u003eCytokinins (CKs) also play a role in germination. Exogenous 6-BA inhibited barley germination, while endogenous trans-zeatin (tZ) levels increased in both varieties. The higher expression of LOG in Stirling suggests its involvement in CK biosynthesis, which may inhibit germination under certain conditions (Song et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Jasmonates promote germination of dormant wheat kernels, and that methyl jasmonate inhibits the expression of the ABA biosynthesis gene (\u003cem\u003eTaNCED1\u003c/em\u003e), leading to a decrease in the ABA content prior to embryo germination (Jacobsen et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Jasmonic acid (JA) showed opposing effects on germination, promoting it in Stirling while inhibiting it in Harrington. Transcriptomic data revealed high expression of JA biosynthesis genes, including \u003cem\u003eLOX\u003c/em\u003e, \u003cem\u003eAOS\u003c/em\u003e, and \u003cem\u003eAOC\u003c/em\u003e, during germination, although JA content decreased over time, potentially due to metabolism.\u003c/p\u003e\u003cp\u003eSA inhibited barley germination, with more pronounced effects in Harrington. Endogenous SA levels decreased during germination, paralleling ABA trends, suggesting SA may act as a negative regulator. Upregulation of SA biosynthesis genes (\u003cem\u003eICS\u003c/em\u003e, \u003cem\u003ePAL\u003c/em\u003e) suggests active synthesis during germination while SA content decreased during germination in both barley varieties, and it may result from degradation or interactions with other hormones likely reduce its content. Under normal growth conditions, SA inhibits barley germination by suppressing GA-induced α-amylase gene expression (Xie et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). SA promotes seed germination of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e under salt stress by reducing oxidative damage, and also promotes germination rate and germination potential of wheat and oat seeds under chromium stress (Lee et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Wang et al., 2024a; Wang et al., 2024b). SA is synthesized through two pathways, ICS and PAL (Peng et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and the genes encoding these synthetic enzymes (\u003cem\u003eICS\u003c/em\u003e, \u003cem\u003eEDS\u003c/em\u003e, \u003cem\u003eEPS\u003c/em\u003e, and \u003cem\u003ePAL\u003c/em\u003e) were all up-regulated during seed germination, and \u003cem\u003eICS\u003c/em\u003e and \u003cem\u003ePAL\u003c/em\u003e were high expression in two barley varieties.\u003c/p\u003e\u003cp\u003eThese findings offer insights into the physiological and molecular differences between fast- and slow-germinating barley varieties. Exogenous hormone treatments demonstrated that GA and ETH promote germination, while ABA, IAA, 6-BA, and SA inhibit it. JA inhibited germination in Harrington but promoted it in Stirling. Differences in endogenous ABA, GA1, IAA, and tZ levels may underlie these variations. Transcriptomic analyses identified 54 candidate genes, including \u003cem\u003eNCED\u003c/em\u003e, \u003cem\u003eAO1\u003c/em\u003e, \u003cem\u003ePP2C\u003c/em\u003e, \u003cem\u003ePYR1\u003c/em\u003e, \u003cem\u003eKAO1\u003c/em\u003e, \u003cem\u003eGA3ox2\u003c/em\u003e, \u003cem\u003eDELLA\u003c/em\u003e, \u003cem\u003eYUCCA\u003c/em\u003e, \u003cem\u003eLOG\u003c/em\u003e, and others, as key regulators of germination. Pan-genomic analyses revealed 19 candidate genes with significant PAVs, which may contribute to genetic differentiation in germination traits. These findings provide a foundation for future studies on the molecular regulation of barley germination and the development of PHS-resistant barley varieties through molecular breeding. These candidate genes could be used to create novel functional alleles by fine-tuning gene expression and introduce the optimal allelic combination into elite cultivars using cutting-edge gene-editing technology.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eABA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAbscisic acid\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eACO\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eACC oxidase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eACS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eACC synthase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eARF\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAuxin response factor\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eAO\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAbscisic Aldehyde oxidase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eAOS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAllene oxide synthase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eAUX\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAuxin\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eBR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eBrassinolide\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCAR5\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eDeveloping grain (5 DAP)\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCAR15\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eDeveloping grain (15 DAP)\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCK\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCytokinin\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCKX\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCytokinin Oxidase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCPS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eent-Copalyl diphosphate synthase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDELLA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eDELLA protein\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDET2\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ede-etiolated 2/steroid 5-alpha-reductase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDMAPP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eDimethylallyl pyrophosphate\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eEDS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eEnhanced disease susceptibility\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eEPS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eEnhanced pseudomonas susceptibility\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eETH\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eEthylene\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eETR2\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eEthylene receptor 2\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eFPKM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eFragments Per Kilobase of transcript per Million mapped reads\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGibberellin\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGI\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGermination index\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGermination rate\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGR24\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e24-hour germination rate\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGR48\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e48-hour germination rate\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGH3\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAcylamide synthetase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGID1\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGibberellin Insensitive Dwarf 1\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eIAA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eIndole-3-acetic acid\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eICS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eIsochorismate synthase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eIPA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eIndole pyruvic acid\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eJA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eJasmonic acid\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eLOX\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eLipoxygenase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eLOG\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eLonely Guy (cytokinin nucleoside 5\u0026prime;-monophosphoribose hydrolase)\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eNCED\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e9-cis-Epoxycarotenoid dioxygenase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eNPR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eNonexpresser of pathogenesis-related genes\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eOPR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e12-Oxophytodienoic acid reductase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePAL\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ePhenylalanine ammonia-lyase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePAVs\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ePresence/Absence variants\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePHS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ePre-harvest sprouting\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePP2C\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003egroup A type 2C protein phosphatase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePYR1\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ePyrabactin resistance 1\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eSalicylic acid\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eS-AdoMets\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eS-adenosylmethionine synthetase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSNPs\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eSingle nucleotide polymorphisms\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003etZ\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003etrans-Zeatin\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTIR/AFB\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eTransport inhibitor response 1/Auxin signaling F-box proteins\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eYUCCA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eFlavin-containing monooxygenase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eZEP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eZeaxanthin epoxidase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study did not involve human participants or animals, and thus, ethics approval and consent to participate were not applicable. All experiments were conducted using plant materials (barley seeds), and the research complied with relevant institutional and national guidelines for plant research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have approved the final manuscript for publication and agree to its submission to the specified journal.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data supporting the findings of this study are available in the manuscript and supplementary material.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work is supported by the program provided by the Engineering Research Center of Ecology and Agricultural Use of Wetland, Ministry of Education KFT202302 and the funding from the National Natural Science Foundation of China (32372052).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthers Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExperimental design:\u0026nbsp;Jun Wu, Bing Zhou, Bo Li, Yanhao Xu, Le Xu.\u003c/p\u003e\n\u003cp\u003eData generation, analysis and curation:\u0026nbsp;Jun Wu, Bing Zhou, Peng Wang, Liya Luo, Ying Zhang. Project supervision: Le Xu. Manuscript draft: Jun Wu, Jiexin Zheng, Bo Li, Xu Le. Final manuscript: Yanhao Xu, Le Xu.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work is supported by the program provided by the Engineering Research Center of Ecology and Agricultural Use of Wetland, Ministry of Education KFT202302 and the funding from the National Natural Science Foundation of China (32372052).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBetts, N.S., Dockter, C., Berkowitz, O., Collins, H., Hooi, M., Lu, Q. et al. 2020. Transcriptional and biochemical analyses of gibberellin expression and content in germinated barley grain. J Exp Bot. 71, 1870-1884. DOI: 10.1093/jxb/erz546.\u003c/li\u003e\n\u003cli\u003eBonnardeaux, Y., Li, C., Lance, R., Zhang, X., Sivasithamparam, K., Appels, R. 2008. 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DOI: 10.1038/s41467-018-07920-5.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6858206/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6858206/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eUnder adverse conditions such as continuous rainfall or high humidity, mature grains are prone to pre-harvest sprouting (PHS) on the parent plant, significantly impacting crop yield and quality. While abscisic acid (ABA) and gibberellin (GA) are well-known regulators of seed germination, the molecular mechanisms by which other phytohormones influence this process remain underexplored. A germination test of 236 barley varieties showed a germination index range of 5.22 to 97.17, indicating extensive genetic diversity. Hormone treatments on fast-germinating Harrington and slow-germinating Stirling revealed that ABA, cytokinin, and salicylic acid inhibited germination, whereas GA and ethylene promoted it. Endogenous hormone quantification indicated that variations in PHS resistance between Harrington and Stirling were primarily due to differences in ABA, GA, auxin, and cytokinin levels. Transcriptomic analysis of barley embryonic tissues identified 260 hormone-responsive differentially expressed genes, with 54 of these genes showing specificity in their expression during germination. Comparative resequencing data from two pairs barley varieties exhibiting contrasting germination traits revealed 19 genes with substantial genetic variation in their coding regions, implicating these genes in the regulation of barley germination properties.\u003c/p\u003e","manuscriptTitle":"Molecular mechanism of phytohormones regulating dormancy and germination of barley seeds","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-15 15:07:26","doi":"10.21203/rs.3.rs-6858206/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"39e26c15-a43a-4886-8620-b524de27a991","owner":[],"postedDate":"July 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-12T12:23:45+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-15 15:07:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6858206","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6858206","identity":"rs-6858206","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}