biostudies-arrayexpressMarzena Małgorzata KurowskaHordeum vulgare subsp. vulgarehttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-14769Drought stress can damage crop growth and lead to a decline in yield, thereby affecting food security, especially in regions vulnerable to climate change. SNAC1 (Stress-responsive NAC1), the NAC transcription factor family member, plays a crucial role in stomatal movement regulation. Effective regulation of stomatal movement is essential for protecting plants from water loss during adverse conditions. Our hypothesis revolves around altering HvSNAC1 activity by introducing a point mutation in its encoding gene, thereby influencing stomatal dynamics in barley. Two TILLING mutants, each harboring missense mutations in the NAC domain, exhibited higher stomatal density after drought stress compared to the parent cultivar 'Sebastian'. These mutants also demonstrated distinct patterns of ABA-induced stomatal movement compared to the ’Sebastian’. To delve deeper, we conducted a comprehensive analysis of the transcriptomes of these mutants and the parent cultivar 'Sebastian' under both optimal watering conditions and ten days of drought stress treatment. We identified differentially expressed genes (DEGs) between the mutants and ‘Sebastian’ under control and drought conditions. Furthermore, we pinpointed DEGs specifically expressed in both mutants under drought conditions. Our experiments revealed that the cis-regulatory motif CACG, previously identified in Arabidopsis and rice, is recognized by HvSNAC1 in vitro. Enrichment analysis led to the identification of the cell wall organization category and potential target genes, such as HvEXPA8 (Expansin 8), HvXTH (Xyloglucan endotransglucosylase/hydrolase), and HvPAE9 (Pectin acetylesterase 9), suggesting their regulation by HvSNAC1. These findings suggest that HvSNAC1 may play a role in regulating genes associated with stomatal density, size, and reopening.biostudies-arrayexpressLibrary Construction - Stranded cDNA library preparation (poly-A selection) was used.Sample Collection - The sample is a fragment of a barley leaf, collected from plants during the experiment. Leaves were excised using sterile scissors to avoid contamination and immediately frozen in liquid nitrogen to preserve RNA integrity. Samples were stored at -80°C until further processing.Nucleic Acid Extraction - The RNAqueous™ Total RNA Isolation Kit (ThermoFisher Scientific) was used per the manufacturer protocol, but instead of a lysis buffer, a TriPure Isolation Reagent (Roche Life Science) was applied.Sequencing - Sequencing of 40M 2x150PE (paired-end PE) reads =12Gb per sample raw data in fastq were performed in Novogene Company (Hong Kong). For reads mapping, barley genome IBSC_V2 was applied (MorexV2_pseudomolecules_assembly; Mascher et al., 2017; https://nov2020-plants.ensembl.org/Hordeum_vulgare/Info/Index).Growth Protocol - To evaluate the impact of specific mutations in the HvSNAC1 gene on the stress response, both mutants and the parent variety 'Sebastian', which is wild type (WT) for mutants, underwent drought treatment using a protocol extensively detailed by Daszkowska-Golec et al. (2017). The drought treatment involved planting barley seeds in pots filled with a mixture of sandy loam, with sand (w/w; 7:2), and supplemented with a nutrient medium. Initially, barley seeds were germinated in Petri dishes containing water-soaked vermiculite and incubated in darkness at 4°C for two days. Subsequently, the germinated seedlings were transferred to a greenhouse for an additional two days before being transplanted into the soil, with 15 seedlings per pot. One pot was one biological replicate, and three biological replicates were tested for each genotype and condition. Soil moisture levels were monitored daily throughout the experiment using Time-domain reflectometer (TDR) EasyTest equipment provided by the Institute of Agrophysics, Polish Academy of Sciences, Poland. The drought assay comprised of two main phases: 1. Control Growth (CG): Seedlings were grown under optimal conditions with soil moisture at 12% vwc for ten days after transplantation. 2. Drought Stress (DS): Seedlings were subjected to severe drought stress, first soil moisture was gradually reduced from 12 to 3% vwc over four days for adaptation to water deficit (AWD), then soil moisture was maintained at 3-1.5% vwc for ten days. The entire drought test spanned 24 days. During the CG and AWD, seedlings were cultivated in a greenhouse at 20˚C/18˚C with a 16/8 hour photoperiod and 400 μEm-2s-1 light intensity from high pressure sodium lamps. After AWD, seedlings were transferred to a growth chamber with a temperature regime of 25˚C/20˚C, maintaining the same photoperiod and light intensity as in the greenhouse.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - Raw sequencing data were subjected to quality control using FastQC to assess read quality and detect the presence or absence of adapter sequences. Reads passing quality thresholds were aligned to the barley genome (IBSC_V2 assembly, MorexV2_pseudomolecules_assembly; Mascher et al., 2017; https://nov2020-plants.ensembl.org/Hordeum_vulgare/Info/Index) using HISAT2. Gene expression counts were normalized and transformed using the limma-voom method. Differential expression analysis was performed with the following thresholds: adjusted p-value ≤ 0.01 and log2 fold change ≤ -1 or ≥ 1 (equivalent to a fold change of 2).MetabolomicsUnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq 6000RNA-seq of coding RNAHordeum vulgare subsp. vulgareMarzena Małgorzata KurowskafalseRNA-seq of hvsnac1.e (E) and Sebastian parent variety (S) during control (C) and drought stress (D)Drought stress can damage crop growth and lead to a decline in yield, thereby affecting food security, especially in regions vulnerable to climate change. SNAC1 (Stress-responsive NAC1), the NAC transcription factor family member, plays a crucial role in stomatal movement regulation. Effective regulation of stomatal movement is essential for protecting plants from water loss during adverse conditions. Our hypothesis revolves around altering HvSNAC1 activity by introducing a point mutation in its encoding gene, thereby influencing stomatal dynamics in barley. Two TILLING mutants, each harboring missense mutations in the NAC domain, exhibited higher stomatal density after drought stress compared to the parent cultivar 'Sebastian'. These mutants also demonstrated distinct patterns of ABA-induced stomatal movement compared to the ’Sebastian’. To delve deeper, we conducted a comprehensive analysis of the transcriptomes of these mutants and the parent cultivar 'Sebastian' under both optimal watering conditions and ten days of drought stress treatment. We identified differentially expressed genes (DEGs) between the mutants and ‘Sebastian’ under control and drought conditions. Furthermore, we pinpointed DEGs specifically expressed in both mutants under drought conditions. Our experiments revealed that the cis-regulatory motif CACG, previously identified in Arabidopsis and rice, is recognized by HvSNAC1 in vitro. Enrichment analysis led to the identification of the cell wall organization category and potential target genes, such as HvEXPA8 (Expansin 8), HvXTH (Xyloglucan endotransglucosylase/hydrolase), and HvPAE9 (Pectin acetylesterase 9), suggesting their regulation by HvSNAC1. These findings suggest that HvSNAC1 may play a role in regulating genes associated with stomatal density, size, and reopening.2025-02-12T00:00:00Z2025-01-21T16:11:29.075Z2025-01-21T16:11:29.075ZE-MTAB-14769ERP168524EFO_0002944EFO_0004170EFO_0003789EFO_0005518EFO_0003816EFO_0003738EFO_0004184