biostudies-arrayexpressKilian DuijtsArabidopsis thalianahttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-16552Seedling establishment is a critical developmental transition that contributes to survival in the local environment. While light is the primary cue driving de-etiolation, how abiotic stress shapes early development in darkness remains unclear. We show that salinity induces a photomorphogenic-like response in dark-grown Arabidopsis thaliana seedlings, disrupting apical hook formation and soil emergence. This experiment provides an organ specific transcriptome of seedlings showing this response.biostudies-arrayexpressSequencing - RNA poly-A enrichment library preparation and sequencing were carried out by Novogene using the illumina NovaSeq 6000 platform.Library Construction - Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers followed by the second strand cDNA synthesis. The library was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification.Growth Protocol - Seeds were surface-sterilized in 0.05% Triton X-100 for 5 min, then in 1:1 household bleach:0.05% Triton X-100 for 10 min, and rinsed four times with sterile water. Sterilized seeds were sown on 50 µM nylon mesh (Sefar BV) over half-strength MS medium (Duchefa) with 388 0.8% plant agar (Duchefa), pH 5.7. After 4 d stratification at 4 °C in darkness, seeds were exposed to ~125 μmol m⁻² s⁻¹ white light at 21 °C for 1 h , then wrapped in foil and grown vertically in darkness at 21 °C for 23 h. Seedlings were subsequently transferred under safe green light to new 0.5× MS plates with or without treatments (see below) and grown in darkness for 48 h unless otherwise stated.Sample Collection - Three day old etiolated seedlings were grown as described above. Dissections were carried out under dark 466 conditions with a green safe-light. Etiolated seedlings were rinsed in a 1:1 solution of RNA-later (invitrogen) 467 and RNAse free water, before separating cotyledons, apical hooks, and hypocotyls (Fig S2A) with a scalpel. 468 Dissected organs were then placed in pre-cooled 2ml safelock Eppendorf tubes containing two 1/8” steel 469 ball bearings (Weldtite, 3906141), and flash frozen in liquid nitrogen.Sample Treatment - For salt stress experiments, 50 mM or 75 mM of NaCl (Fluka), KCl (Amresco), NaNO3 (Sigma), KNO3 (Sigma), and MgCl2 (Sigma) were added to 0.5 MS media. For osmotic stress experiments, 100 mM or 150 mM of sorbitol was added to 0.5 MS media. Stock solutions of N-1-naphthylphthalamic acid (NPA; Supelco) were made by dissolving directly in dimethyl sulfoxide (DMSO; Sigma) to reach a stock concentration of 10 mM, which was later added to media to achieve a working concentration of 5 µM. Equal volumes of DMSO (0.05%) were added to 0.5 MS as mock treatment.Nucleic Acid Extraction - Each tube contained organs from 30- 470 45 seedlings, and were ground frozen for 30s at 25 Hz twice. RNA-isolation was carried out using the RNEasy 471 Plant Mini kit (Qiagen) with on-column DNAse treatment, in accordance with manufacturer instructions. 472 Three samples were combined during addition of RLT bumer, whereby the bumer was transferred from one 473 tube to the next, until the lysate of 90-125 seedlings was suspended in the bumer to be loaded into the 474 QIAshredder spin columnOrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesSequence Alignment - Read quality checking, trimming, mapping and quantification against the TAIR10 genome (66) were conducted using a NextFlow nf-core/rna-seq pipeline v3.9Data Transformation - Normalization to obtain tpm and scaled values were performed in nextflow pipeline/rnaseq v. 3.9MetabolomicsUnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq 6000Seedling establishment is highly sensitive to environmental cues, with light serving as a principal regulator. In darkness, seedlings enter skotomorphogenesis, marked by hypocotyl elongation and apical hook formation, which helps seedlings emerge from the soil to reach the light. Here, we show that salinity impairs soil emergence of Arabidopsis thaliana seedlings by inducing a partially photomorphogenic like phenotype in darkness, characterized by reduced apical hook curvature. Within the hook, salt stress diminished differential epidermal cell elongation required to drive hook bending, and reduced both the auxin signalling maximum and PIN3 abundance on the concave side of the hook. Transcriptome analysis revealed that salt and osmotic stresses directly alter organ-specific transcriptional profiles, including the NaCl-mediated repression of B-BOX DOMAIN PROTEIN 28 ( BBX28 ) in hooks and cotyledons. Under control conditions, BBX28 protein accumulated asymmetrically across the hook, but this pattern was lost under salinity. Notably, the ubiquitin ligase CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) exhibited an opposite gradient to BBX28, which was similarly disrupted by salt stress or by the inhibition of polar auxin transport, presenting auxin as an upstream regulator of COP1 spatial distribution. Together, these findings establish COP1 asymmetry as a novel feature of the apical hook, indicating that spatial regulation—not just absolute levels—shapes COP1 function, and reveal how salinity disrupts hormonal, transcriptional and protein networks to compromise seedling establishment under stress. <h4>Significance Statement</h4> Seedling establishment is a critical developmental transition that contributes to survival in the local environment. While light is the primary cue driving de-etiolation, how abiotic stress shapes early development in darkness remains unclear. We show that salinity induces a photomorphogenic-like response in dark-grown Arabidopsis thaliana seedlings, disrupting apical hook formation and soil emergence. This response involves disrupted asymmetrical epidermal cell elongation and auxin signalling, repression of B-BOX DOMAIN PROTEIN 28 ( BBX28 ), and loss of a previously unrecognized spatial gradient of the key light regulator COP1 across the apical hook. Our discovery that COP1 is spatially regulated and disrupted by stress, reveals a new dimension of light signalling and developmental control, highlighting how environmental stress constrains seedling establishment.RNA-seq of coding RNAArabidopsis thalianaSalt stress disrupts local auxin and COP1 gradients in Arabidopsis apical hooksElizabeth van VeenKilian DuijtsElizabeth van Veen, Jesse J. Küpers, Xizheng Chen, Yu Him Tang, Thijs de Zeeuw, Kilian Duijts, Scott Hayes, Christa Testerink, Charlotte M. M. GommersCharlotte GommersfalseRNAseq of dark dissected, 3 day old Arabidopsis (col0) seedlings, including cotyledons, apical hooks and hypocotylsSeedling establishment is a critical developmental transition that contributes to survival in the local environment. While light is the primary cue driving de-etiolation, how abiotic stress shapes early development in darkness remains unclear. We show that salinity induces a photomorphogenic-like response in dark-grown Arabidopsis thaliana seedlings, disrupting apical hook formation and soil emergence. This experiment provides an organ specific transcriptome of seedlings showing this response.2026-06-23T00:00:00Z2026-06-23T08:41:53.667Z2026-01-15T13:50:48.195ZE-MTAB-16552ERP187616EFO_0002944EFO_0004170EFO_0003789EFO_0004917EFO_0005518EFO_0003816EFO_0003738EFO_0004184EFO_000396910.64898/2025.12.03.691840