biostudies-arrayexpressGareth GillardPicea abieshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15297Conifers such as Picea abies (Norway spruce) are among the most radiosensitive plant species, yet the molecular impact of ionising radiation on their stem cells remains poorly understood. This study examines the transcriptomic response of embryogenic stem cells to gamma radiation, focusing on mechanisms that influence stem cell viability and maintenance of stemness. Clonal callus cultures were generated in vitro and exposed to chronic gamma radiation at dose rates of 1, 10, 20, 40, and 100 mGy h⁻¹ for 144 hours. Total RNA was extracted at the end of the exposure period from both irradiated and control samples. Strand-specific RNA sequencing was performed to quantify dose-dependent changes in gene expression. The dataset provides a resource for studying molecular responses to ionising radiation in conifers, with particular relevance to DNA damage repair, oxidative stress responses, chromatin modification, protein homeostasis, and hormone signalling pathways. This work contributes to understanding how ecologically and economically important tree species may respond to environmental radiation exposure at the cellular level.biostudies-arrayexpressGrowth Protocol - Embryogenic callus cultures of Picea abies (line ID#B10W) were maintained in vitro on proliferation medium (PM) composed of AL basal medium with inositol, modified vitamin mix, 10 μM 2,4-D, 5 μM BA, 1% sucrose, and 0.3% Phytagel. Cultures were grown in 9 cm Petri dishes at 23 °C in darkness prior to gamma irradiation.Sample Treatment - In vitro callus cultures were exposed to continuous gamma radiation for 144 hours at dose rates of 0 (control), 1, 10, 20, 40, or 100 mGy/h using a ⁶⁰Co source at the FIGARO low dose irradiation facility (NMBU). Petri dishes were positioned at calibrated distances and rotated midway to ensure even exposure. Controls were kept shielded from radiation in the same room.Nucleic Acid Extraction - Total RNA was extracted using the MasterPure Complete DNA and RNA Purification Kit (Epicentre) following the manufacturer’s protocol, with modifications: 1–2% polyvinylpyrrolidone (PVP, mw 360,000) was added to the extraction buffer, and 1,4-dithiothreitol (DTT) was replaced with 3 μl beta-mercaptoethanol. RNA was quantified using a NanoDrop ND-1000 and assessed for quality using an Agilent 2100 Bioanalyzer.Sample Collection - Embryogenic callus cultures of Picea abies (line B10W), derived from a single zygotic embryo, were maintained in vitro on proliferation medium under dark conditions at 23 ± 1°C. Callus aggregates were exposed to continuous gamma radiation from a ⁶⁰Co source at specified dose rates (0–100 mGy/h) for 144 hours. At the end of exposure, callus samples were harvested, flash-frozen in liquid nitrogen, and stored at −80°C prior to RNA extraction.Sequencing - Libraries were sequenced at the Norwegian Sequencing Centre (University of Oslo) using an Illumina HiSeq 4000 instrument. Strand-specific libraries were sequenced in paired-end mode with 150 bp read length, across two lanes.Library Construction - Strand-specific RNA-seq libraries were prepared using the TruSeq stranded mRNA library preparation protocol (Illumina) according to the manufacturer’s instructions. Libraries were constructed from total RNA and sequenced as 150 bp paired-end reads.OrganizationMINSEQE ScoreAssays and DataMAGE-TAB FilesUnknownTranscriptomicsGenomicsProteomicsIllumina HiSeq 4000Conifers are among the most radiosensitive plant species. Elevated, sublethal levels of ionising radiation result in reduced apical dominance in conifers, indicating a negative effect on shoot apical meristems (SAMs). The SAMs, harbouring the pluripotent stem cells, generate all the cells of the shoot, enabling growth and reproduction. However, knowledge on the effects of ionising radiation on such stem cells is scarce, but important for risk assessment and radioprotection of plants in contaminated ecosystems. Here, we assessed the sensitivity of in vitro -grown stem cells of Norway spruce to 144-h of gamma irradiation at 1-100 mGy h -1 , using such cells as a model for molecular toxicity of gamma radiation in conifers. Although there were no visible effects of the gamma irradiation on cell proliferation and subsequent embryo formation, dose rate-dependent DNA damage was observed at ≥10 mGy h -1 , and comprehensive organelle damage at all dose rates. Massive dose rate-dependent transcriptome changes occurred, with downregulation of a range of genes related to cell division, DNA repair and protein folding but upregulation of stress-related hormonal pathways and several antioxidant-related genes. The upregulation of such genes, survival and continued proliferation of at least a subset of cells and the post-irradiation normalisation of expression of DNA repair and protein-folding genes together with somatic embryo formation suggest that stem cells are able to recover from gamma-irradiation-induced stress. Collectively, regardless of cellular abnormalities after gamma irradiation, and huge transcriptomic shifts towards stress management pathways, the pluripotent stem cell cultures were able to retain their stemness. <h4>Highlights</h4> Norway spruce stem cells retained stemness after gamma irradiation (1-100 mGy h -1 ). DNA damage was observed at ≥ 10 mGy h -1 and organelle damage at all dose rates. Huge dose rate-dependent transcriptomic changes occurred after 144-h irradiation. Downregulated key DNA repair-genes recovered post-irradiation. Induction of stress-management and antioxidant genes may aid stem cell maintenance.RNA-seq of coding RNAPicea abiesGamma radiation-induced molecular toxicity and effects on pluripotent stem cells of the radiosensitive conifer Norway spruce (Picea abies)Payel Bhattacharjee, YeonKyeong Lee, Marcos Viejo, Gareth B Gillard, Simen Rød Sandve, Torgeir R. Hvidsten, Brit Salbu, Dag A Brede, Jorunn E OlsenJorunn OlsenPayel BhattacharjeeGareth GillardYeonKyeong LeefalseRNA-seq of Norwegian spruce embryos exposed to gamma radiationConifers such as Picea abies (Norway spruce) are among the most radiosensitive plant species, yet the molecular impact of ionising radiation on their stem cells remains poorly understood. This study examines the transcriptomic response of embryogenic stem cells to gamma radiation, focusing on mechanisms that influence stem cell viability and maintenance of stemness. Clonal callus cultures were generated in vitro and exposed to chronic gamma radiation at dose rates of 1, 10, 20, 40, and 100 mGy h⁻¹ for 144 hours. Total RNA was extracted at the end of the exposure period from both irradiated and control samples. Strand-specific RNA sequencing was performed to quantify dose-dependent changes in gene expression. The dataset provides a resource for studying molecular responses to ionising radiation in conifers, with particular relevance to DNA damage repair, oxidative stress responses, chromatin modification, protein homeostasis, and hormone signalling pathways. This work contributes to understanding how ecologically and economically important tree species may respond to environmental radiation exposure at the cellular level.2025-07-14T00:00:00Z2025-07-01T18:33:39.843Z2025-07-01T18:33:39.843ZE-MTAB-15297ERP174397EFO_0002944EFO_0004170EFO_0003789EFO_0005518EFO_0003738EFO_0004184EFO_000396910.1101/2025.06.02.653021