Temporal regulation of gene expression during auxin-triggered crown root formation in barley: an integrated approach

Plant and Cell Physiology, 2025, 66(9),1284–1303. https://doi.org/10.1093/pcp/pcaf077, Advance Access publication on 13 July 2025, available online at https://academic.oup.com/pcp © The Author(s) 2025. Published by Oxford University Press on behalf of the Japanese Society of Plant Physiologists. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Editor-in-Chief’s Choice Regular Paper Temporal regulation of gene expression during auxin-triggered crown root formation in barley: an integrated approach Nikola Koˇrínková1,2,Alexie Techer1,Maria Majeská ˇ Cudejková1,Dieu Thu Nguyen1,David Kopeˇcný3, Bruno Contreras-Moreira 4,Pavla Navrátilová5,Pascal Gantet 1,6and Véronique Bergougnoux 1,* 1Czech Advanced Technology and Research Institute (CATRIN), Palacký University Olomouc, ˇ Slechtitel˚u 241/27, Olomouc 779 00, Czech Republic, 2Department of Biochemistry, Faculty of Science, Palacký University Olomouc, ˇ Slechtitel˚u 241/27, Olomouc 779 00, Czech Republic, 3Department of Experimental Biology, Faculty of Science, Palacký University Olomouc, 17. listopadu 1192/12, Olomouc 779 00, Czech Republic, 4Computational and Structural Biology, EEAD-CSIC, Av. Montañana 1.005, Zaragoza 50059, Spain, 5Centre of Plant Structural and Functional Genomics, Institute of Experimental Botany, Academy of Sciences, ˇ Slechtitel˚u 893/31, Olomouc 779 00, Czech Republic, 6UMR DIADE, Université de Montpellier, IRD, CIRAD, Avenue Agropolis 911, Montpellier 34398, France *Corresponding author: E-mail, [email protected] Received 6 March 2025; Accepted 29 June 2025 Abstract Cereal plants possess a fibrous root system in which crown roots form the major component. Crown roots develop post-embryonically from the lower, mostly underground nodes of the stem base. A strict spatiotemporal regulation of gene expression governs this process. Much of the knowledge about signaling pathways controlling crown root initiation (CRI) and development comes from rice. However, distinct regulatory mechanisms may have evolved in other cereals to adapt to different habitats. In this study, using a Crown Root Inducible System (CRIS), we aimed to investigate the early molecular regulation of barley CRI. We revealed dynamic transcriptomic changes within the first 24 hours following auxin stimulation. Among the differentially expressed genes, we identified orthologs of important CRI regulators from other cereals, demonstrating that CRIS is suitable for uncovering genes involved in CRI. Further, ATAC-seq revealed that CRI relies on changes in chromatin accessibility near root development-related genes and within distal intergenic regions. Finally, we focused on two transcription factors, HvNAC013 and CBF12C, which likely play roles in both CRI and abiotic stresses. By performing DAP-seq, we determined their genome-wide binding sites and identified their potential downstream targets. Data suggest that CBF12C is a putative target of HvNAC013, along with other auxin-responsive genes implicated in CRI. We propose that HvNAC013 and CBF12C function as part of a transcription factor network involved in CRI and potentially modulate root architecture in response to environmental conditions. This study enhances our understanding of the CRI mechanism in barley. Keywords: ATAC-seq •auxin •Barley •crown roots • DAP-seq •transcriptomics Introduction Barley (Hordeum vulgare L.), a cereal crop belonging to the Triticeae tribe, ranks fourth among cereals in global cultivation area (FAO 2024). In recent years, barley has become a model organismforcerealcropstudiesduetoitsadvantageouscharacteristics, including self-pollination, diploid genome of moderate size and a low chromosome number (Von Bothmer et al. 2003). Advances in molecular tools, availability of mutant populations, large collections of landraces and wild accessions have fueled growing interest in functional genomics of barley (Russell et al. 2016,Szurman-Zubrzycka et al. 2018,Navrátilová et al. 2022). Barley’s adaptability to diverse environmental conditions has proven especially valuable for research on mechanisms of abiotic stress tolerance (Gürel et al. 2016). Roots play a critical role in nutrient and water acquisition, anchorage, and plant-microbe interactions, making them a potential breeding target (Den Herder et al. 2010). One of the characteristic features of monocot plants, including cereals, is their fibrous root system. Although at the seedling stage the root system is composed of primary and seminal roots, later, the mature root system is essentially made of post-embryonic roots emerging from the stem, called crown roots (CR) (Hackett 1968). CR emerge from a subset of pericycle-like cells in the ground meristem of the stem base (Itoh et al. 2005,Coudert et al. 2013). Their initiation is primarily regulated by auxin and cytokinin that function antagonistically in the early stage of primordia establishment (Itoh et al. 2005,Zhao et al. 2009). The local accumulation of auxin serves as a positional signal for the emergence of a new root (Benková et al. 2003). The asymmetric auxin distribution is maintained by polar auxin transport, which Downloaded from https://academic.oup.com/pcp/article/66/9/1284/8198101 by Palacky University user on 16 October 2025 Auxin-induced crown root initiation in barley directs auxin to specific cells in the ground meristem (Kitomi et al. 2008). The proper positioning of the auxin efflux carrier PIN-FORMED 1 (PIN1) is mediated by the rice (Oryza sativa) CROWN ROOTLESS 4 (CRL4) gene, encoding a membraneassociated guanine-nucleotide exchange factor of the ADPribosylation factor G (GNOM) protein (Steinmann et al. 1999, Geldner et al. 2003,Kitomi et al. 2008). Auxin perception triggers a cascade of molecular events that lead to crown root initiation (CRI) (Inukai et al. 2005). Cytokinin signaling is involved both in initiation and in tissue differentiation during the development of CR primordia (Neogy et al. 2021,Omary et al. 2022). The initiation of a new organ formation requires fine-tuning of gene expression. This process entails the binding of transcription factors to specific DNA sequences, located within regulatory regions, thereby activating or repressing transcription. The study of transcriptional regulators implicated in CRI initiated approximately two decades ago with the rice crl1 mutant (Inukai et al. 2005,Liu et al. 2005). This mutant lacks CR in early development and the mature plants develop only afewCR.TheCRL1 gene encodes a transcription factor of theASYMMETRICLEAVES2(AS2)/LATERAL ORGAN BOUNDARIES (LOB) DOMAIN (LBD) protein family, and its expression is directly regulated by auxin through the AUXIN/INDOLE3-ACETIC ACID(AUX/IAA)-AUXIN RESPONSE FACTOR (ARF) signaling pathway (Inukai et al. 2005;Liu et al. 2005). The observation that the adult crl1 mutant plants still develop few CR suggests the existence of a CRL1-independent pathway (Inukai et al. 2005). Since these early studies, a complex network of signaling pathways regulating CRI, CR outgrowth and emergence was proposed in the rice model (Meng et al. 2019,Tanaka et al. 2023,Chennakesavulu et al. 2024). Rootdevelopmentisregulated by hormones that respond to environmental stimuli, resulting in root plasticity (Zhou et al. 2021,Kang et al. 2022,Kim et al. 2022). Transcription factors are key to the transduction of this signal. The NO APICAL MERISTEM (NAM), ARABIDOPSIS TRANSCRIPTION ACTIVATION FACTOR (ATAF), and CUP-SHAPED COTYLEDON (CUC) (NAC) family acts in developmental, abiotic and biotic stress pathways (Han et al. 2023). Until now, 167 NAC genes have been identified in barley (Shen et al. 2009,Murozuka et al. 2018). However, only few of them have been functionally described (Kjaersgaard et al. 2011,Christiansen et al. 2016, Chen et al. 2023). The plant-specific transcription factor of the APETALA 2/ETHYLENE RESPONSIVE FACTOR (AP2/ERF) superfamily is involved in development and adaptive response to environment (Xie et al. 2019). In barley, AP2/ERF comprises 185 genes, divided into 3 families based on their domain composition (Sakuma et al. 2002,Ding et al. 2021). Tolerance to abiotic stresses is typically associated with members of the C-REPEAT BINDING FACTOR/DEHYDRATION RESPONSIVE ELEMENT BINDING 1 (CBF/DREB1) group (Stockinger et al. 1997,Yang et al. 2020). In rice, CRL1 regulates several NAC and AP2/ERF members, suggesting their involvement in CR formation (Coudert et al. 2015,Lavarenne et al. 2019). It appears that NAC and AP2/ERF transcription factors represent potential targets for improving both root architecture and abiotic stress tolerance in crops (Erpen et al. 2018). The current study aimed to elucidate the genome-wide responses of barley seedlings during auxin-induced CR formation. We investigated the temporal transcriptomic changes within the first 24 h post-induction of CRI. We identified major transcriptional programs activated in the early phases of CRI and analyzed the involvement of chromatin accessibility in this process. Furthermore, we examined the possible role of HvNAC013 and CBF12C transcription factors in CR formation in barley. We identified potential targets of HvNAC013 and CBF12C, as well as the specific cis-regulatory elements recognized by these transcription factors. Based on our findings, we propose that HvNAC013 and CBF12C might function both in CR development and response to abiotic stress. Results and Discussion Crown root inducible system allows deciphering the dynamic transcriptomic changes controlling early crown root formation The mature barley embryo contains two axillary meristem (AXM) primordia, one in the axil of the coleoptile and the other in the axil of the first leaf primordium; these primordia will later develop into tillers (Hussien et al. 2014). Primary and seminal root primordia appear from the basal pole of the embryo, and are protected by the coleorhiza (Jackson 1922,Hackett 1968, Aloni and Griffith 1991,Rossini et al. 2018). The first CR is formed within 3 days after germination (Nguyen et al. 2024), and 5 to 10 days after germination, the first CR emerge from the stem base (Hackett 1968,Knipfer and Fricke 2011). The unpredictable timing of CRI challenges a detailed molecular investigation. Therefore, to study the early events of CR primordium formation, we adapted the Lateral Root (LR) Inducible System (LRIS) that has been used in Arabidopsis thaliana, maize (Zea mays), Medicago truncatula and rice to study gene regulation of LR initiation (Jansen et al. 2013, Crombez et al. 2016,Motte et al. 2023). The Crown Root Inducible System (CRIS), similarly to LRIS, is based on the primary inhibition of CR by an inhibitor of the polar auxin transport (N-(1-naphthyl)phthalamic acid; NPA), followed by the induction by a synthetic auxin. In this system, the formation and emergence of seminal roots and tillers was not affected (Supplemental Fig. S1, S2), in agreement with previous reports (Woodward and Marshall 1988,Agusti and Greb 2013,Riaz et al. 2023). Barley seedlings did not develop any CR primordium when maintained in NPA for 8 days (Fig. 1a). The auxin treatment stimulated the synchronous initiation of few CR primordia (Fig. 1b-d). The CR primordia were observed 36 h after auxin induction (hai) (Fig. 1b, c) when they appeared as a mass of dividing cells. From 48 hai, we observed dome-shaped CR primordia (Fig. 1d). When seedlings were transferred from CRIS to large hydroponics with 1/2Hoagland for three weeks, seedlings that were treated with auxin for 24 h developed significantly higher number of CR than 1285 Downloaded from https://academic.oup.com/pcp/article/66/9/1284/8198101 by Palacky University user on 16 October 2025 Koˇrínková et al. Figure 1. Cross-sections of stem base of barley seedlings grown in the crown root inducible system (CRIS). Seedlings were grown for 8 days in the presence of NPA, an inhibitor of polar auxin transport. Stem bases were collected before treatment with auxin (a) and at different times after auxin treatment: 24 h (b), 36 h (c), and 48 h (d). Cross sections (4 μm thick) were stained with periodic acid–Schiff and Naphthol blue black. Pictures were acquired with a Zeiss microscope with a 10x objective. Bar = 200 μm; asterisk stands for crown root primordium; sr, seminal root; t, tiller; vb, vascular bundles. the control plants (Supplemental Fig. S2). Further, our repeated histological studies revealed that auxin treatment consistently and repeatably induced the synchronous initiation of up to 6 CR primordia. Our results show that CRIS efficiently induced CR formation and suggest that CR priming and initiation occur within 36 hai. Therefore, we restricted our study to the first 24 h to identify the early molecular events of CRI. We performed the whole transcriptome analysis of the stem base of barley seedlings collected before induction andat3,6,9,and24hai(Supplementary Table S1A-S1D). Quality control parameters of the RNA-seq data are shown in Supplementary Fig. S3 and Supplementary Table S2A. Genes known to regulate the tiller number in barley and other cereals were not differentially expressed in our data, highlighting the suitability of the system to study genes related to CRI (Supplementary Table S1E). The number of significantly upregulated genes (padj <0.05) ranged from 406 (3 hai) to 936 (6 hai) (Fig. 2a).Thenumberofsignificantly downregulated genes (padj <0.05) varied from 491 (3 hai) to 956 (9 hai) (Fig. 2b). We identified 176 and 107 genes, respectively upand down-regulated across all time points (Supplementary Table S1F). The top genes exhibiting the highest activation included two AUX/IAA auxin signal repressors, HvIAA28 and HvIAA21, not yet associated with CR development. Further, genes encoding proteins involved in phytohormone homeostasis were upregulated. These include two GRETCHEN HAGEN 3 genes (HvGH3–7 and HvGH3–4), which maintain biologically active auxin levels and are homologous to the A. thaliana GH3.3 gene, known to be involved in adventitious rooting (Staswick et al. 2005, Gutierrez et al. 2012). One of the most induced genes encodes a gibberellin 2-beta-dioxygenase (HvGA2ox6a), suggesting that gibberellin deactivation is required for CRI in barley. This is supported by the observation that reduced gibberellin levels promote LR proliferation in poplar and that GA2ox is a downstream target of auxin signaling during CR formation in rice (Gou et al. 2010,Coudert et al. 2015). Genes encoding enzymes involved in the biosynthesis of cytokinins, ethylene and strigolactones were also induced across all time points. In contrast, a pronounced downregulation was observed for genes associatedwithJASMONATEZIM-DOMAIN(JAZ)proteinsand members of the AP2/ERF family (Supplementary Table S1F). 1286 Downloaded from https://academic.oup.com/pcp/article/66/9/1284/8198101 by Palacky University user on 16 October 2025 Auxin-induced crown root initiation in barley Figure 2. Venn diagrams showing overlap of up- (a) and down- (b) regulated genes in barley stem base at 3 h, 6 h, 9 h and 24 h after auxin treatment, (padj <0.05). Diagrams were generated by InteractiVenn (Heberle et al. 2015). JAZ proteins suppress jasmonate signaling and positively affect root growth (Li et al. 2017). To summarize, our data highlight an extensive hormonal crosstalk during auxin-mediated CRI in barley. Genes associated with the auxin response were upregulated across the whole time series (Supplementary Figs. S4–S7, Supplementary Table S3). The initial 3 hai were characterized by an enrichment in transcriptional regulators for both the upand down-regulated genes (Supplementary Fig. S4, Supplementary Table S3A). Biological processes such as ‘shoot and root development’, ‘regulation of hormone levels’ or ‘rhythmic processes’ were also enriched. In A. thaliana, the circadian clock regulates auxin signaling related genes and coordinates LR emergence (Voß et al. 2015). By 6 hai, genes related to ‘cellulose catabolism’ and ‘positive regulation of developmental growth’ were activated, while genes related to ‘cell wall organization and biogenesis’ were downregulated (Supplementary Fig. S5, Supplementary Table S3B). In A. thaliana, swelling of pericycle cells precedes the first division during LR initiation (Vermeer et al. 2014). Cell wall remodeling enzymes are likely required for loosening of the cell wall during this process, as well as for proper positioning of the asymmetric cell divisions (Lewis et al. 2013,Ramakrishna et al. 2019). At 9 hai, ‘acetyltransferases’ and ‘signaling receptor activators’ were specifically enriched, while ‘polyamine biosynthesis’ and ‘responses to abiotic stimuli’ were inhibited(Supplementary Fig. S6,SupplementaryTable S3C). At 24 hai, we observed an enrichment in terms ‘cytokinin riboside 5´-monophosphate phosphoribohydrolase’ (cytokinin biosynthesis) and ‘negative regulation of cell population proliferation’ (Supplementary Fig. S7,Supplementary Table S3D). Conversely, cytokinin degradation and reactive oxygen species (ROS) metabolic processes were inhibited. The importance of ROS homeostasis for root architecture was recently described in rice, wherein a high number of ROS homeostasis-related genes were downregulated during CRI and CR outgrowth (Kumar et al. 2024). Auxin triggers fast upregulation of genes related to root apical meristem development Recently, in barley, we identified HvCRL1 as the ortholog of the rice CRL1 and the maize ROOTLESS CONCERNING CROWN AND SEMINAL ROOTS (RTCS) gene that integrates both auxin and cytokinin to initiate CR formation (Hetz et al. 1996,Inukai et al. 2005,Liu et al. 2005,Taramino et al. 2007). In barley, using the CRIS, HvCRL1 gene was significantly upregulated in the stem base of barley as soon as 1 h after auxin induction and hvcrl1 knock-out harbored lower number of CR (Nguyen et al. 2025). We also characterized HvCRL1L1 as the putative ortholog of the rice DEGENERATED HULL 1 (DH1)/OsLBD16 and the maize LBD24 which functions in CR primordium initiation and outgrowth (Stelpflug et al. 2016,Garg et al. 2023,Geng et al. 2024). In our current dataset, both HvCRL1 and HvCRL1L1 transcripts strongly accumulated in response to auxin, with their expression remaining high during the whole duration of the experiment (Table 1, Supplementary Table S1). The transcript accumulation of these two genes, markers of CRI, confirms that the CRIS is suitable to uncover genes involved in the CRI in barley. Our transcriptomic data revealed that auxin induced rapid changes in the expression of barley genes orthologous to genes controlling root initiation and development in rice (Table 1). The barley QUIESCENT-CENTER SPECIFIC HOMEOBOX (HvQHB) was induced within 3 hai and remained elevated thereafter. In rice, QHB is involved in the specification and maintenance of the root apical meristem stem cells (Kamiya et al. 2003b), suggesting that the root apical meristem stem cells specify fast after auxin induction. In the first 3 hai, genes involved in auxin signaling, homeostasis and transport, such as HvIAA13,HvARF02,HvPIN1A, 1287 Downloaded from https://academic.oup.com/pcp/article/66/9/1284/8198101 by Palacky University user on 16 October 2025 Koˇrínková et al. Table 1. List of barley orthologs of known crown root formation regulators from rice and their expression after auxin treatment Barley Rice LFC (padj <0.05) References Gene name Accession (HORVU.MOREX.r3.) Gene name Accession 3h 6h 9h 24 h HvCRL1 4HG0408280 CRL1 Os03g0149100 2.61 3.16 2.83 3.09 Inukai et al. 2005;Liu et al. 2005;Nguyen et al. 2025 HvCRL1L1 6HG0630410 DH1/OsLBD16 Os02g0820500 2.88 3.89 4.31 4.45 Garg et al. 2023;Geng et al. 2024;Nguyen et al. 2025 HvQHB 3HG0301330 QHB Os01g0854500 1.84 2.68 2.38 2.70 Kamiya et al. 2003b;Coudert et al. 2015;Lavarenne et al. 2019 HvCLE25 6HG0632110 OsCLE206 Os02g0826300 -1.35 1.85 1.89 Huangwei et al. 2013 HvIAA13 3HG0287760 OsIAA6 Os01g0741900 0.79 1.00 0.71 0.70 Lavarenne et al. 2019 HvARF02 7HG0726940 OsARF19 Os06g0702600 0.66 0.90 0.82 0.87 Lavarenne et al. 2019 HvPIN1A 6HG0615550 PIN1A Os02g0743400 0.98 1.49 1.41 1.20 Lavarenne et al. 2019 HvGH3–8 3HG0294170 OsGH3-1 Os01g0785400 1.69 1.28 1.01 1.16 Lavarenne et al. 2019 HvWOX11 2HG0111270 OsWOX11 Os07g0684900 4.04 4.77 4.61 4.28 Zhao et al. 2009;Zhao et al. 2015;Geng et al. 2023,2024 HvCKX4 3HG0318720 OsCKX4 Os01g0940000 2.61 3.03 3.20 3.24 Gao et al. 2014;Geng et al. 2023 HvROP 6HG0602690 OsROP Os04g0561200 -2.95 3.44 3.45 Gonin et al. 2022 HvbHLH044 4HG0406540 OsbHLH044 Os03g0188400 -−0.74 −0.79 -Lavarenne et al. 2019;Gonin et al. 2022 HvERF3 3HG0294880 OsERF3 Os01g0797600 -0.77 0.75 0.42 Zhao et al. 2015 RGF 6HG0574910 RGF Os02g0190700 -1.78 2.61 2.19 Matsuzaki et al. 2010 RGF 2HG0205670 RGF Os04g0643800 -2.46 3.11 2.50 Matsuzaki et al. 2010 HvPLT1 2HG0203350 OsPLT1 Os04g0653600 --0.65 -Garg et al. 2022 HvSCR 2HG0168770 OsSCR2 Os12g0122000 1.21 1.30 1.65 -Kamiya et al. 2003a and HvGH3–8, were upregulated. HvIAA13 is a putative ortholog of the rice OsIAA6, a negative regulator of the CRL1-QHB interaction (Lavarenne et al. 2019). Concurrently, barley genes mediating auxin-cytokinin crosstalk were activated, including orthologs of the WUSCHEL-RELATED HOMEOBOX 11 (OsWOX11), RESPONSE REGULATOR 2 (OsRR2), and CYTOKININ OXIDASE/DEHYDROGENASE 4 (OsCKX4). OsWOX11 integrates auxin and cytokinin signaling pathways and interacts with the CRL1 (Zhao et al. 2009,Geng et al. 2023,2024). This interaction is required for the expression of OsCKX4, which controls cytokinin homeostasis during CRI and CR outgrowth (Gao et al. 2014,Geng et al. 2023). As cytokinin signaling maintains root meristem size by stimulating cell differentiation, during CRI, it must be repressed via cytokinin type-A RRs (Ioio et al. 2007,Kitomi et al. 2011). Six hours after induction, orthologs of two other CRL1 targets were significantly differentially expressed: a Rho GTPase (HvROP) and a basic helix–loop–helix transcription factor (HvbHLH044). HvROP was upregulated, while the HvbHLH044 was transiently downregulated. In rice, both genes promote crown root development (Coudert et al. 2015,Gonin et al. 2022). OsbHLH044 is a repressor of a gene network regulating programmed cell death and senescence during the later stages of root development (Gonin et al. 2022). Additionally, the HvERF3 gene was upregulated. In rice, OsERF3 represses cytokinin signaling by positively regulating OsRR2 during CR initiation, whereas it stimulates cytokinin signaling during CR emergence through its interaction with OsWOX11 to repress OsRR2 (Zhao et al. 2015). A similar regulatory mechanism might operate in barley to control CR formation. Additionally, two genes from the GOLVEN/root meristem growth factor (RGF)/CLAVATA3 (CLV3)/ENDOSPERM SURROUNDING REGION (ESR)-related (CLE)-like family were significantly upregulated. RGFs are essential for proper root growth, as they contribute to the root stem cell niche maintenance (Matsuzaki et al. 2010). HvCLE25, coding a CLE protein, was also found to be upregulated from 6 hai. In rice, the CLE/WOX module regulates root meristem maintenance and vascular tissue development (Huangwei et al. 2013). By 9 hai, barley PLETHORA 1 (HvPLT1) was significantly upregulated,inlinewithOsPLT1 being expressed during rice CRI (Garg et al. 2022). OsPLT1 induces CR formation, likely through activating auxin biosynthesis genes (Garg et al. 2022). Our data reveal that RGF and PLT are sequentially expressed, consistent with described RGF-mediated control of PLT distribution patterns (Matsuzaki et al. 2010). At 9 hai, the expression of HvRR2 returns to its pre-induction state, and by 24 hai it is downregulated, diminishing cytokinin signaling repression. The repression of HvRR2 might involve the ERF3/WOX11 complex, as described in rice (Zhao et al. 2015). Indeed, whereas HvWOX11 is upregulated as soon as 3 hai, HvERF3 expression increases from 6 hai, prior to the repression of HvRR2.Also,barleySCARECROW (HvSCR) gene, significantly upregulated between 3 and 9 hai, was no longer differentially expressed at 24 hai. In rice, OsSCR is specifically expressed in endodermalcells,whereitiscrucialforasymmetriccelldivisions that generate cortex cell layers. Following division, OsSCR is downregulated in the daughter cells (Kamiya et al. 2003a). Our results suggest that in barley the cortex cell lineages are defined between 9 and 24 hai. Chromatin accessibility landscape specific for barley stem base was identified In rice, a profound chromatin remodeling was suggested to occur in the CR primordium founder cells (Lavarenne et al. 2020,Garg et al. 2022). In barley, chromatin remodeling may be crucial during CRI as evidenced by the differential 1288 Downloaded from https://academic.oup.com/pcp/article/66/9/1284/8198101 by Palacky University user on 16 October 2025 Auxin-induced crown root initiation in barley Figure 3. Comparison of ACRs identified in barley stem base at different times following auxin treatment and changes in ATAC-seq signal near the transcription start site (TSS) of differentially expressed genes. (a) Venn diagram illustrating the overlap of ACRs before induction (BI = 0 h) and the different time points (3 h; 6 h; 9 h; 24 h) following auxin treatment. (b) Distribution of ACRs relative to gene features, analyzed by ChIPseeker (Yu et al. 2015,Wang et al. 2022). (c, d) ATAC-seq read density ±1.5 kb around TSS of significantly up- (c) or down-regulated (d) differentially expressed genes (padj <0.05) after auxin treatment. Statistically significant if P<.01 (paired t-test). expression of genes encoding proteins involved in posttranslational histone modification, nucleosome remodeling, chromatin structure and DNA homeostasis (Supplementary Table S1A–S1D). Using the Assay for Transposase-Accessible Chromatin coupled to sequencing (ATAC-seq), we identified Accessible Chromatin Regions (ACRs) in barley stem base tissue primed for CR development (Buenrostro et al. 2013). Consistent with a high similarity among samples (Supplementary Fig. S8A), the number of ACRs remained relatively stable across all conditions, with 40 962 ATAC-seq peaks before auxin treatment (0 h), 45 760 peaks after 3 h of treatment, 40 145 peaks after 6 h, 36 397 peaks after 9 h, and 39 579 peaks after 24 h (Supplementary Table S2B). The comparative analysis revealed 26 319 peaks common to all time points (Fig. 3a). Only a minor proportion (up to 7.3%) of ACRs was located in unique genomic regions, likely reflecting the use of samples collected from identical tissue in a short time frame. In ATAC-seq, the signal is typically enriched in active genomic regions around the transcription start sites (TSSs), in promoters and enhancers (Buenrostro et al. 2013). Here, we examined the spatial distribution of ACRs. The majority of ACRs were annotated to distal regions (Fig. 3b), with 60% of ATAC-seq peaks located within the 10 kb of a TSS, and half of these within 1 kb (Supplementary Fig. S8B-S8F), in agreement with the previous report (Lu et al. 2019). For our study, we considered a promoter as a region covering −500 bp upstream and +100 bp downstream to the TSS (Pavlu et al. 2024). More than 25% of ACRs were assigned to promoter regions, while ∼13% of them were associated with 3´UTRs (Fig. 3b). TSS regions of upregulated genes displayed significantly higher ATAC-seq signals after auxin treatment (Fig. 3c). The highest increase in chromatin accessibility was observed at 9 h, coinciding with the enrichment of acetyltransferases (SupplementaryTableS3C).Incontrast,nosignificantchangein chromatin accessibility around the TSS (P<.01) was observed for downregulated genes (Fig. 3d). Altogether, our data suggest that, in barley, chromatin remodeling contributes to the activation of genes regulating CR development, whereas transcriptional inactivation might rather involve transcriptional 1289 Downloaded from https://academic.oup.com/pcp/article/66/9/1284/8198101 by Palacky University user on 16 October 2025 Koˇrínková et al. Figure 4. Comparison of differentially accessible regions (DARs) identified in barley stem base at different time points following auxin treatment and their distribution in the genome. (a) Venn diagram illustrating the overlap of DARs at different time points (3 h; 6 h; 9 h; 24 h) following auxin treatment. (b) Distribution of DARs relative to gene features, analyzed by ChIPseeker (Yu et al. 2015;Wang et al. 2022). (c-e) examples of DAR positions in the genome relative to selected differentially expressed genes potentially involved in CrI. ATAC-seq signal distribution at 0 h and 3 h after auxin treatment. Rectangles represent accessible chromatin regions (ACRs) at 0 h and 3 h, DARs, or genes. Genome visualizations in (c-e) were generated using the integrative genomics viewer (Robinson et al. 2011). repressors, DNA methylation, or other non-coding elements (Zhang et al. 2006,Barrett et al. 2012). Changes in chromatin accessibility are important for controlling gene expression during CRI Cis-regulatory elements are key determinants of the tight spatiotemporal gene regulation that determines cell and tissue fate specification during plant development (Weber et al. 2016). Genomic sites that undergo significant changes in chromatin accessibility are termed Differentially Accessible Regions (DARs) and represent potentially essential cisregulatory elements. Given the high similarity of ATAC-seq data across our experimental conditions, only a small proportion of ATAC-seq peaks were characterized as DARs (logCPM >−2, FDR <0.15; Supplementary Tables S2C and S4). Only 21 DARs were recognized at 3 hai while a maximum of 459 sites were identified at 6 hai (Supplementary Tables S2C and S4). Most DARs were specific to their respective time points (Fig. 4a)and were located in distal intergenic regions (Fig. 4b). Distal ACRs are proposed to act as transcriptional enhancers controlling gene expression while interacting with their target genes via chromatin looping (Lu et al. 2019,Ricci et al. 2019). We focused on DARs located within the genic and proximal regions, excluding ‘distal intergenic’ annotations. Between 17 to 50% of DARs were associated with DEGs (Supplementary Table S2C). A notable alteration in the chromatin accessibility was detected in the 3´-UTR region of HvWOX11 as 1290 Downloaded from https://academic.oup.com/pcp/article/66/9/1284/8198101 by Palacky University user on 16 October 2025 Auxin-induced crown root initiation in barley early as 3 hai (Fig. 4c). Later on, DARs were also observed in the promoter and distal upstream region of HvWOX11 (Supplementary Table S4B). Recently, Geng et al. (2024) showed that OsWOX11 binds the histone demethylase JMJ706 to remove histone H3 lysine 9 (H3K9me2) mark from the OsLBD16 promoter, enabling OsLBD16 expression. Similarly, OsWOX11 recruits the ALTERATION/DEFICIENCY IN ACTIVATION 2/GENERAL CONTROL NON-DEPRESSIVE histone acetyltransferase complex to regulate genes involved in auxin transport, cell wall biosynthesis, and energy metabolism, all required for cell proliferation in the developing root meristem (Zhou et al. 2017). Altogether, it suggests that HvWOX11 expression is regulated at the chromatin level. Assuming that the function of WOX11 is conserved among cereals, HvWOX11 might in turn recruit chromatin modifying enzymes to regulate downstream genes during CRI (Zhou et al. 2017,Geng et al. 2024). The ATAC-seq signal spanning ∼3.5 kb over HvIAA28 at 3 hai (Fig. 4d) correlated with its expression pattern (Supplementary Table S1A). Later on, chromatin accessibility increased in the regulatory regions of other auxinresponsive genes, such as different AUX/IAAs,HvGH3–4,orFbox encoding genes (Supplementary Fig. S9A, Supplementary Table S4). Similar regulatory patterns were observed across many other genes including LONELY GUY (LOG), HvGA2ox6a (Supplementary Fig. S9B), a putative JmjC domain-containing histone demethylase (Supplementary Fig. S9C), and a gene encoding a member of the PUMILIO family of RNA-binding proteins, which is implicated in shoot stem cell maintenance androotcellproliferation(SupplementaryFig.S9D;Francischini and Quaggio 2009;Huang et al. 2014). Early downregulation at the chromatin level was noted in the 3´-UTR region of the barley REGULATOR OF CHROMOSOME CONDENSATION 1 (HvRCC1), correlating with its decreased expression (Fig. 4e). Later, downregulated DARs were identified near EXORDIUM-like 1 (HvEXL1)(Supplementary Fig. S9E), or a gene encoding a cell wall restructuring enzyme, xyloglucan endotransglucosylase/hydrolase (XTH) (Supplementary Fig. S9F). Downregulated DARs spanned genomic regions enriched in transcription factor binding motifs, which thus became less accessible after auxin induction. Surprisingly, in two conditions (6 hai and 9 hai), the number of recognized transcription factor binding motifs was considerably higher in downregulated DARs compared to upregulated DARs (Supplementary Table S5). Additionally, DNA-binding motifs specific to certain transcription factor families, such as ERFs and AGAMOUS-LIKE, were more frequent in the downregulated DARs. Compared to the transcriptomic changes during CRI, chromatin accessibility alterations appear less dynamic. This might reflect generally lower sensitivity of DARs detection, or suggests that the transcriptional regulation of the early stages of CRI relies on qualitative recruitment of key genomic sites, rather than widespread quantitative changes in chromatin accessibility. HvNAC013 and CBF12C show distinct expression patterns during CRI To gain insights into the molecular cascade leading to CR formation, we focused on barley orthologs of the rice OsNAC39 (Os03g0327100) and OsERF28 (Os08g0545500), which are direct targets of CRL1 with a possible role in CR development. OsNAC39 is a transcription factor of the NAC family, which is part of the CRL1-dependent gene regulatory network (Coudert et al. 2015,Lavarenne et al. 2019). Beyond its role in CR formation, it is upregulated under drought and salinity stresses and by abscisic acid (Jeong et al. 2010,Lima et al. 2015,Ahn et al. 2017,Neogy et al. 2019). OsERF28 is upregulated in a CRL1dependent manner during the early stage of CR formation, but also in roots in response to drought, acetic acid, jasmonate and abscisic acid, suggesting a role in drought avoidance through root plasticity (Oh et al. 2009,Lavarenne et al. 2019,Ogawa et al. 2021). In line with previous phylogenetic analyses, we identified HvNAC013 astheorthologofOsNAC39 (SupplementaryFig.S10; Christiansen et al. 2011;Murozuka et al. 2018).Theroleof HvNAC013 in CR development was recently suggested in barley seedlings grown in normal condition (Nguyen et al. 2024). Our data indicate that HvNAC013 is steadily expressed across all time points following auxin treatment, which was validated by qPCR (Fig. 5a). Chromatin accessibility in its promoter region did not change significantly over the time (Fig. 5c). Consistent with the auxin-independent expression pattern of HvNAC013, auxin response elements were absent in the −3kb and +100 bp region of TSS. Instead, we identified cis-elements responsive to other phytohormones, light or low temperature (Supplementary Table S6A). Furthermore, the TSS region of HvNAC013 was screened for the typical LBD binding motif, LBD-box (GCGGCG), and for the recently described CRL1-box (CACA[A/C]C) (Husbands et al. 2007,Gonin et al. 2022). An LBD-box was identified 43 bp downstream of a start codon, and the CRL1-box was represented twice (2 kb and 3 kb from TSS) (Fig. 5e). It is unlikely that HvNAC013 is a direct target of HvCRL1. Indeed, LBD transcription factors function as homodimers or heterodimers, preferentially binding two DNA motifs separated by a spacer sequence, following mechanism identical to the molecular caliper described for ARF transcription factors (Korasick et al. 2015,Lee et al. 2017,Chen et al. 2019). Concerning OsERF28, 10 potential barley orthologs were identified (Supplementary Fig. S11). OsERF28, also called OsDREB1I, is a member of the CBF3 subgroup of the CBF/ DREB1 subfamily (Skinner et al. 2005,Moon et al. 2019). Therefore, we narrowed down the selection to 7 barley genes belonging to the same phylogenetic group (Skinner et al. 2005, Pasquariello et al. 2014). CBF12C was the only member of the CBF3 subgroup that was upregulated after auxin treatment (Supplementary Table S7). In contrast to HvNAC013,both transcriptomic and qPCR analyses showed that the expression of CBF12C was induced as early as 3 hai, reaching a maximum at6hai(Fig. 5b,Supplementary Table S1A–S1D). Chromatin 1291 Downloaded from https://academic.oup.com/pcp/article/66/9/1284/8198101 by Palacky University user on 16 October 2025 Koˇrínková et al. Figure 5. Gene expression of HvNAC013 and CBF12C and the position and accessibility of cis-regulatory elements in their promoters. (a, b) gene expression of HvNAC013 (a) and CBF12C (b) in barley stem base at different time points after auxin treatment, relative to the stem base before auxin treatment (0 h). Presented data show the mean ±standard error of the mean of three biological replicates. The lowercase letters indicate statistically significant differences between groups (one-way ANOVA followed by Tukey’s multiple comparison of mean rank, considered significantly different if padj <0.05, analyzed in GraphPad prism 8.0.1). Groups sharing the same letter are not significantly different. (c, d) ATACseq read density ±0.5 kb around transcription start site (TSS) of HvNAC013 (c) and CBF12C (d). TSS regions are shown in opposite orientations, as the genes lie on different strands. Differentially accessible regions at 6 hai is marked by ∗. (e, f) position of LBD-box (GCGGCG) and CRL1-box (CACA[a/C]C) 3 kb upstream to 100 bp downstream of HvNAC013 TSS (e) and CBF12C TSS (f). accessibility in the promoter region of CBF12C increased transiently between 3 and 6 hai and this region was recognized as a significant DAR (Fig. 5d,Supplementary Table S4B). We identified two auxin responsive elements in the ∼2.5 kb upstream region of the TSS (Supplementary Table S6B). Other cis-regulatory elements were associated with response to drought, light, methyl jasmonate, abscisic acid, or salicylic acid. In addition, the analysis revealed four dehydration-responsive elements (DREs), involved in responses to low temperature and water deficit, which are recognized by AP2/ERF transcription factors (Stockinger et al. 1997,Liu et al. 1998). Additionally, we discovered several LBD-box and CRL1-box elements in the −3kband+100 bp region of TSS (Fig. 5f). The spacing of these motifs would likely allow dimerization of LBDs, as some are separated by only between 3 and 26 bp (Chen et al. 2019). In brief, our data suggest that CBF12C might be (i) a direct target of CRL1 and (ii) regulated by a transcription factor of the AP2/ERF family. 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