DNA damage response (DDR) is an essential mechanism by which living organisms maintain their genomic stability. In plants, DDR is important also for normal growth and yield. Here, we explored the DDR of a temperate model crop barley (Hordeum vulgare) at the phenotypic, physiological, and transcriptomic levels. By a series of in vitro DNA damage assays using the DNA strand break (DNA-SB) inducing agent zeocin, we showed reduced root growth and expansion of the differentiated zone to the root tip. Genome-wide transcriptional profiling of barley wild-type and plants mutated in DDR signaling kinase ATAXIA TELANGIECTASIA MUTATED AND RAD3-RELATED (hvatr.g) revealed zeocin-dependent, ATR-dependent, and zeocin-dependent/ATR-independent transcriptional responses. Transcriptional changes were scored also using the newly developed catalog of 421 barley DDR genes with the phylogenetically-resolved relationships of barley SUPRESSOR OF GAMMA 1 (SOG1) and SOG1-LIKE (SGL) genes. Zeocin caused up-regulation of specific DDR factors and down-regulation of cell cycle and histone genes, mostly in an ATR-independent manner. The ATR dependency was obvious for some factors associated with DDR during DNA replication and for many genes without an obvious connection to DDR. This provided molecular insight into the response to DNA-SB induction in the large and complex barley genome.

Centre of Plant Structural and Functional Genomics Institute of Experimental Botany of the Czech Academy of Sciences Olomouc Czechia

Department of Cell Biology and Genetics Faculty of Science Palacký University Olomouc Czechia

Institute of Biology Biotechnology and Environmental Protection Faculty of Natural Sciences University of Silesia in Katowice Katowice Poland

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Ciccia A, Elledge SJ. The DNA damage response: Making it safe to play with knives. Mol. Cell. 2010;40:179–204. doi: 10.1016/j.molcel.2010.09.019. PubMed DOI PMC

Tibbetts RS, Abraham RT. PI3K-related kinases. In: Gutkind JS, editor. Signaling Networks and Cell Cycle Control: The Molecular Basis of Cancer and Other Diseases. Humana Press; 2000. pp. 267–301.

Garcia V, et al. AtATM is essential for meiosis and the somatic response to DNA damage in plants. Plant Cell. 2003;15:119–132. doi: 10.1105/tpc.006577. PubMed DOI PMC

Culligan K, Tissier A, Britt A. ATR regulates a G2-phase cell-cycle checkpoint in Arabidopsis thaliana. Plant Cell. 2004;16:1091–1104. doi: 10.1105/tpc.018903. PubMed DOI PMC

Helton ES, Chen X. p53 modulation of the DNA damage response. J. Cell. Biochem. 2007;100:883–896. doi: 10.1002/jcb.21091. PubMed DOI

Yoshiyama KO. SOG1: A master regulator of the DNA damage response in plants. Genes Genet. Syst. 2016;90:209–216. doi: 10.1266/ggs.15-00011. PubMed DOI

Bourbousse C, Vegesna N, Law JA. SOG1 activator and MYB3R repressors regulate a complex DNA damage network in Arabidopsis. Proc. Natl. Acad. Sci. U. S. A. 2018;115:E12453–E12462. doi: 10.1073/pnas.1810582115. PubMed DOI PMC

Ogita N, et al. Identifying the target genes of SUPPRESSOR OF GAMMA RESPONSE 1, a master transcription factor controlling DNA damage response in Arabidopsis. Plant J. 2018;94:439–453. doi: 10.1111/tpj.13866. PubMed DOI

Velemínský J, Zadražil S, Pokorný V, Gichner T, Švachulová J. Repair of single-strand breaks and fate of N-7-methylguanine in DNA during the recovery from genetical damage induced by N-methyl-N-nitrosourea in barley seeds. Mutat. Res. Mol. Mech. Mutagen. 1973;17:49–58. doi: 10.1016/0027-5107(73)90252-2. DOI

Velemínský J, Zadražil S, Pokorný V, Gichner T. DNA repair synthesis stimulated by mutagenic N-methyl-N-nitrosourea in barley seeds and free embryos. Mutat. Res. Mol. Mech. Mutagen. 1977;44:43–51. doi: 10.1016/0027-5107(77)90113-0. DOI

Velemínský J, Pokorný V, Šatava J, Gichner T. Post-replication DNA repair in barley embryos treated with N-methyl-N-nitrosourea. Mutat. Res. Mol. Mech. Mutagen. 1980;71:91–99. doi: 10.1016/0027-5107(80)90009-3. DOI

Lupu A, Nevo E, Zamorzaeva I, Korol A. Ecological–genetic feedback in DNA repair in wild barley, Hordeum spontaneum. Genetica. 2006;127:121–132. doi: 10.1007/s10709-005-2611-0. PubMed DOI

Vu GTH, et al. Repair of site-specific DNA double-strand breaks in barley occurs via diverse pathways primarily involving the sister chromatid. Plant Cell. 2014;26:2156–2167. doi: 10.1105/tpc.114.126607. PubMed DOI PMC

Gruszka D, Marzec M, Szarejko I. The barley EST DNA replication and repair database (bEST-DRRD) as a tool for the identification of the genes involved in DNA replication and repair. BMC Plant Biol. 2012;12:88. doi: 10.1186/1471-2229-12-88. PubMed DOI PMC

Volkova PY, et al. Early response of barley embryos to low- and high-dose gamma irradiation of seeds triggers changes in the transcriptional profile and an increase in hydrogen peroxide content in seedlings. J. Agron. Crop Sci. 2020;206:277–295. doi: 10.1111/jac.12381. DOI

Szurman-Zubrzycka ME, et al. HorTILLUS—A rich and renewable source of induced mutations for forward/reverse genetics and pre-breeding programs in barley (Hordeum vulgare L.) Front. Plant Sci. 2018;9:216. doi: 10.3389/fpls.2018.00216. PubMed DOI PMC

Jaskowiak J, et al. Al-tolerant barley mutant hvatr.g shows the ATR-regulated DNA damage response to maleic acid hydrazide. Int. J. Mol. Sci. 2020;21:8500. doi: 10.3390/ijms21228500. PubMed DOI PMC

Povirk LF, Wübter W, Köhnlein W, Hutchinson F. DNA double-strand breaks and alkali-labile bonds produced by bleomycin. Nucleic Acids Res. 1977;4:3573. doi: 10.1093/nar/4.10.3573. PubMed DOI PMC

Hu Z, Cools T, De Veylder L. Mechanisms used by plants to cope with DNA damage. Annu. Rev. Plant Biol. 2016;67:439–462. doi: 10.1146/annurev-arplant-043015-111902. PubMed DOI

Nowicka A, et al. Dynamics of endoreduplication in developing barley seeds. J. Exp. Bot. 2021;72:268–282. doi: 10.1093/jxb/eraa453. PubMed DOI

Szurman-Zubrzycka M, et al. ATR, a DNA damage signaling kinase, is involved in aluminum response in barley. Front. Plant Sci. 2019;10:1299. doi: 10.3389/fpls.2019.01299. PubMed DOI PMC

Bouyer D, et al. Genome-wide identification of RETINOBLASTOMA RELATED 1 binding sites in Arabidopsis reveals novel DNA damage regulators. PLOS Genet. 2018;14:e1007797. doi: 10.1371/journal.pgen.1007797. PubMed DOI PMC

Anderson HJ, et al. Arabidopsis thaliana Y-family DNA polymerase η catalyses translesion synthesis and interacts functionally with PCNA2. Plant J. 2008;55:895–908. doi: 10.1111/j.1365-313X.2008.03562.x. PubMed DOI

Murozuka E, Massange-Sánchez JA, Nielsen K, Gregersen PL, Braumann I. Genome wide characterization of barley NAC transcription factors enables the identification of grain-specific transcription factors exclusive for the Poaceae family of monocotyledonous plants. PLoS ONE. 2018;13:e0209769. doi: 10.1371/journal.pone.0209769. PubMed DOI PMC

Gorbatova IV, et al. Studying gene expression in irradiated barley cultivars: PM19L-like and CML31-like expression as possible determinants of radiation hormesis effect. Agronomy. 2020;10:1837. doi: 10.3390/agronomy10111837. DOI

Takahashi N, et al. A regulatory module controlling stress-induced cell cycle arrest in Arabidopsis. eLife. 2019;8:e43944. doi: 10.7554/eLife.43944. PubMed DOI PMC

Povirk LF. DNA damage and mutagenesis by radiomimetic DNA-cleaving agents: Bleomycin, neocarzinostatin and other enediynes. Mutat. Res. Mol. Mech. Mutagen. 1996;355:71–89. doi: 10.1016/0027-5107(96)00023-1. PubMed DOI

Adé J, Belzile F, Philippe H, Doutriaux M-P. Four mismatch repair paralogues coexist in Arabidopsis thaliana: AtMSH2, AtMSH3, AtMSH6-1 and AtMSH6-2. Mol. Gen. Genet. MGG. 1999;262:239–249. PubMed

Mizuno D, et al. Three nicotianamine synthase genes isolated from maize are differentially regulated by iron nutritional status. Plant Physiol. 2003;132:1989–1997. doi: 10.1104/pp.102.019869. PubMed DOI PMC

Lee D-K, et al. The rice OsNAC6 transcription factor orchestrates multiple molecular mechanisms involving root structural adaptions and nicotianamine biosynthesis for drought tolerance. Plant Biotechnol. J. 2017;15:754–764. doi: 10.1111/pbi.12673. PubMed DOI PMC

Kobbe D, Blanck S, Focke M, Puchta H. Biochemical characterization of AtRECQ3 reveals significant differences relative to other RecQ helicases. Plant Physiol. 2009;151:1658–1666. doi: 10.1104/pp.109.144709. PubMed DOI PMC

Garg P, Stith CM, Sabouri N, Johansson E, Burgers PM. Idling by DNA polymerase δ maintains a ligatable nick during lagging-strand DNA replication. Genes Dev. 2004;18:2764–2773. doi: 10.1101/gad.1252304. PubMed DOI PMC

Pan T, et al. A novel WEE1 pathway for replication stress responses. Nat. Plants. 2021;7:209–218. doi: 10.1038/s41477-021-00855-8. PubMed DOI

Vega-Muñoz I, Herrera-Estrella A, Martínez-de la Vega O, Heil M. ATM and ATR, two central players of the DNA damage response, are involved in the induction of systemic acquired resistance by extracellular DNA, but not the plant wound response. Front. Immunol. 2023;14:2385. doi: 10.3389/fimmu.2023.1175786. PubMed DOI PMC

Povirk LF, Houlgrave CW. Effect of apurinic/apyrimidinic endonucleases and polyamines on DNA treated with bleomycin and neocarzinostatin: specific formation and cleavage of closely opposed lesions in complementary strands. Biochemistry. 1988;27:3850–3857. doi: 10.1021/bi00410a049. PubMed DOI

Chen J, Ghorai MK, Kenney G, Stubbe J. Mechanistic studies on bleomycin-mediated DNA damage: multiple binding modes can result in double-stranded DNA cleavage. Nucleic Acids Res. 2008;36:3781–3790. doi: 10.1093/nar/gkn302. PubMed DOI PMC

Shimada K, et al. TORC2 signaling pathway guarantees genome stability in the face of DNA strand breaks. Mol. Cell. 2013;51:829–839. doi: 10.1016/j.molcel.2013.08.019. PubMed DOI

Georgieva M, Stoilov L. Assessment of DNA strand breaks induced by bleomycin in barley by the comet assay. Environ. Mol. Mutagen. 2008;49:381–387. doi: 10.1002/em.20396. PubMed DOI

Mahapatra K, Roy S. SOG1 transcription factor promotes the onset of endoreduplication under salinity stress in Arabidopsis. Sci. Rep. 2021;11:11659. doi: 10.1038/s41598-021-91293-1. PubMed DOI PMC

Chen H, et al. The ATR–WEE1 kinase module promotes SUPPRESSOR OF GAMMA RESPONSE 1 translation to activate replication stress responses. Plant Cell. 2023 doi: 10.1093/plcell/koad126. PubMed DOI PMC

Robinson H, et al. Genomic regions influencing seminal root traits in barley. Plant Genome. 2016 doi: 10.3835/plantgenome2015.03.0012. PubMed DOI

Abdel-Ghani AH, et al. Genome-wide association mapping in a diverse spring barley collection reveals the presence of QTL hotspots and candidate genes for root and shoot architecture traits at seedling stage. BMC Plant Biol. 2019;19:216. doi: 10.1186/s12870-019-1828-5. PubMed DOI PMC

Kilian J, et al. The AtGenExpress global stress expression data set: protocols, evaluation and model data analysis of UV-B light, drought and cold stress responses. Plant J. 2007;50:347–363. doi: 10.1111/j.1365-313X.2007.03052.x. PubMed DOI

Hecht SM. Bleomycin: New perspectives on the mechanism of action. J. Nat. Prod. 2000;63:158–168. doi: 10.1021/np990549f. PubMed DOI

Chankova SG, Dimova E, Dimitrova M, Bryant PE. Induction of DNA double-strand breaks by zeocin in Chlamydomonas reinhardtii and the role of increased DNA double-strand breaks rejoining in the formation of an adaptive response. Radiat. Environ. Biophys. 2007;46:409–416. doi: 10.1007/s00411-007-0123-2. PubMed DOI

Wagner U, Edwards R, Dixon DP, Mauch F. Probing the diversity of the Arabidopsis glutathione S-transferase gene family. Plant Mol. Biol. 2002;49:515–532. doi: 10.1023/A:1015557300450. PubMed DOI

Chong J, et al. Downregulation of a pathogen-responsive tobacco UDP-Glc:phenylpropanoid glucosyltransferase reduces scopoletin glucoside accumulation, enhances oxidative stress, and weakens virus resistance. Plant Cell. 2002;14:1093–1107. doi: 10.1105/tpc.010436. PubMed DOI PMC

Simon C, et al. The secondary metabolism glycosyltransferases UGT73B3 and UGT73B5 are components of redox status in resistance of Arabidopsis to Pseudomonas syringae pv. tomato. Plant Cell Environ. 2014;37:1114–1129. doi: 10.1111/pce.12221. PubMed DOI

Preuss SB, Britt AB. A DNA-damage-induced cell cycle checkpoint in Arabidopsis. Genetics. 2003;164:323–334. doi: 10.1093/genetics/164.1.323. PubMed DOI PMC

Friesner JD, Liu B, Culligan K, Britt AB. Ionizing radiation–dependent γ-H2AX focus formation requires ataxia telangiectasia mutated and ataxia telangiectasia mutated and Rad3-related. Mol. Biol. Cell. 2005;16:2566–2576. doi: 10.1091/mbc.e04-10-0890. PubMed DOI PMC

Yelagandula R, et al. The histone variant H2A.W defines heterochromatin and promotes chromatin condensation in Arabidopsis. Cell. 2014;158:98–109. doi: 10.1016/j.cell.2014.06.006. PubMed DOI PMC

Veylder LD, Larkin JC, Schnittger A. Molecular control and function of endoreplication in development and physiology. Trends Plant Sci. 2011;16:624–634. doi: 10.1016/j.tplants.2011.07.001. PubMed DOI

Hase Y, Trung KH, Matsunaga T, Tanaka A. A mutation in the UVI4 gene promotes progression of endo-reduplication and confers increased tolerance towards ultraviolet B light. Plant J. 2006;46:317–326. doi: 10.1111/j.1365-313X.2006.02696.x. PubMed DOI

Kumar N, Larkin JC. Why do plants need so many cyclin-dependent kinase inhibitors? Plant Signal. Behav. 2017;12:e1282021. doi: 10.1080/15592324.2017.1282021. PubMed DOI PMC

Lang, L. et al. The DREAM complex represses growth in response to DNA damage in Arabidopsis. Life Sci. Alliance4, (2021). PubMed PMC

Farmer LM, et al. The RAD23 family provides an essential connection between the 26S proteasome and ubiquitylated proteins in Arabidopsis. Plant Cell. 2010;22:124–142. doi: 10.1105/tpc.109.072660. PubMed DOI PMC

Bègue H, Mounier A, Rosnoblet C, Wendehenne D. Toward the understanding of the role of CDC48, a major component of the protein quality control, in plant immunity. Plant Sci. 2019;279:34–44. doi: 10.1016/j.plantsci.2018.10.029. PubMed DOI

Culligan KM, Robertson CE, Foreman J, Doerner P, Britt AB. ATR and ATM play both distinct and additive roles in response to ionizing radiation. Plant J. 2006;48:947–961. doi: 10.1111/j.1365-313X.2006.02931.x. PubMed DOI

Lobet G, Pagès L, Draye X. A novel image-analysis toolbox enabling quantitative analysis of root system architecture. Plant Physiol. 2011;157:29–39. doi: 10.1104/pp.111.179895. PubMed DOI PMC

Coiro M, Truernit E. Xylem characterization using improved pseudo-Schiff propidium iodide staining of whole mount samples and confocal laser-scanning microscopy. In: de Lucas M, Etchhells JP, editors. Xylem: Methods and Protocols. Springer; 2017. pp. 127–132. PubMed

Mascher, M. Pseudomolecules and annotation of the third version of the reference genome sequence assembly of barley cv. Morex [Morex V3]. e.10.5447/ipk/2021/3. (2021).

Kim D, Paggi JM, Park C, Bennett C, Salzberg SL. Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nat. Biotechnol. 2019;37:907–915. doi: 10.1038/s41587-019-0201-4. PubMed DOI PMC

Liao Y, Smyth GK, Shi W. The Subread aligner: Fast, accurate and scalable read mapping by seed-and-vote. Nucleic Acids Res. 2013;41:e108. doi: 10.1093/nar/gkt214. PubMed DOI PMC

Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15:550. doi: 10.1186/s13059-014-0550-8. PubMed DOI PMC

Camacho C, et al. BLAST+: architecture and applications. BMC Bioinformatics. 2009;10:421. doi: 10.1186/1471-2105-10-421. PubMed DOI PMC

Mascher M, et al. Long-read sequence assembly: A technical evaluation in barley. Plant Cell. 2021;33:1888–1906. doi: 10.1093/plcell/koab077. PubMed DOI PMC