Integrated transcriptomic and biochemical analyses reveal mechanisms of drought adaptation in strawberry (Fragaria × ananassa L.).

Drought is a major abiotic stress that limits strawberry productivity worldwide, yet the underlying physiological and molecular mechanisms of cultivar-specific tolerance remains poorly understood. This study integrated physiological, biochemical, and transcriptomic analyses to dissect drought responses of two contrasting cultivars of Fragaria × ananassa: drought-tolerant 'Kara' and drought-sensitive 'E22'. Plants were exposed to both progressive water-deficit (Def) and PEG-induced osmotic stress under controlled greenhouse conditions. Physiologically, the two cultivars diverged substantially: E22 exhibited progressive chlorophyll decline and severe reduction in PSII efficiency (Fv/Fm dropping to 0.64 under PEG), whereas Kara maintained and even increased its chlorophyll content throughout the experiment while sustaining higher Fv/Fm values (> 0.72). Proline accumulation - a key osmoprotective response - was significantly higher in Kara (1.50 vs 0.78 µmol g⁻1 FW under PEG at peak stress). Transcriptomic profiling (18 RNA-Seq libraries, ~ 872 million reads) revealed fundamentally different stress strategies. E22 showed extensive transcriptome reprogramming, with 22,558 differentially expressed transcripts (DETs) under PEG, including down-regulation of photosynthesis-related pathways and up-regulation of ABA signaling, MAPK cascades, and heat shock response. In contrast, Kara exhibited a restrained response (< 900 DETs per stress condition), with differential expression focused on homeostasis-related functions such as transcription regulation, protein folding, and RNA processing. PEG-induced osmotic stress consistently elicited a stronger transcriptomic response than progressive water deficit in both cultivars, suggesting that osmotic sensing is a primary driver of large-scale gene regulation under drought. Enrichment analyses (GO/KEGG via ORA and GSEA) confirmed that E22 activated canonical drought and heat stress pathways, while Kara selectively engaged transcriptional regulators and RNA-processing genes. Transcription factor profiling showed extensive activation of AP2/ERF, NAC, WRKY, and MYB families in E22, with far fewer TFs altered in Kara. qRT-PCR validation of selected DEGs corroborated the RNA-Seq findings. These results provide new insights into cultivar-specific drought adaptation and identify candidate genes for breeding stress-resilient strawberry varieties.