Tissue-specific response in seedling stage reveals key physio-biochemical and molecular network associated with drought tolerance in cucumber

• First comprehensive study on tissue-specific expression of cucumber under drought stress. • Leaf and root tissues expressed more sensitivity to important traits. • CsSOD, CsCAT and CsAPX were upregulated in tolerant genotypes under stress. • Transcription factors like Cs1G589140 , and Cs1G008430 were upregulated in tolerant genotypes. • Provide insight into tissue-specific modulation for future crop designing. Cucumber, a key warm-season vegetable crop, is significantly affected by water stress. To better understand drought tolerance in cucumber and its mechanism, a drought tolerance matrix was calculated for 109 genotypes based on root system architecture (RSA) under moisture stress. Based on these seedlings from six diverse genotypes, representing tolerant, susceptible, and intermediate drought response groups, were analyzed for tissue-specific physio-biochemical traits under osmotic stress. Further, one genotype from each group underwent tissue-specific qRT-PCR analysis across different stress durations to study molecular networks and stress regulation at the tissue level.DGC-21 and WBC-23-2 which are drought tolerant and with the highest drought tolerance matrix score, showed very low root diameter and less reduction of total root length and root surface area. Physio-biochemical parameters like net photosynthesis rate, relative water content (RWC), osmotic potential, membrane stability index (MSI) and proline level were significantly higher in tolerant genotypes. However, lipid peroxidation level and amount of hydrogen peroxide (H 2 O 2 ) were considerably higher in susceptible ones. Antioxidant enzymes generally showed a significantly higher activity in the tolerant genotype, only in leaf. In the drought-tolerant cucumber genotype WBC-23-2, key oxidative stress-related genes (e.g., CsSOD, CsCAT , and CsAPX ) and transcription factors (e.g., Cs1G589140 and Cs1G008430 ) were upregulated in leaf, stem, and root tissues after 48 h of stress, highlighting their essential role in mitigating drought effects. Additionally, the downregulation of auxin-responsive genes and the upregulation of ethylene-responsive transcription factors in tolerant genotypes suggest mechanisms that help prevent seedling drooping under stress. These findings will enhance understanding of drought responses in cucumbers and provide a basis for developing strategies to improve stress resilience.