Proteomic analysis of heat stress response and population diversity in Zygophyllum coccineum using hierarchical clustering and superoxide dismutase as a molecular biomarker

Rising global temperatures and more frequent heatwaves threaten seedling establishment in arid ecosystems, yet the molecular basis of thermal resilience in desert-adapted plants remains poorly understood. This study aimed to assess protein expression and antioxidant responses in Z. coccineum seedlings under heat stress, and to identify conserved and population-specific mechanisms of thermal resilience. Seeds of Z. coccineum from Wadi El-Rayan, Kom Oshim (Fayoum, Egypt), and Al Kharj (Saudi Arabia) were germinated under control (25 °C) and heat stress (45 °C) conditions. Seedlings were harvested after 10 days for protein extraction. Here, we investigated germination, superoxide dismutase (SOD) activity gels, and protein profiles by SDS-PAGE. For proteomics, proteins were digested and analyzed by LC-MS/MS, with label-free quantification (NSAF), normalization, and bioinformatic analyses to identify heat-responsive proteins and population-specific molecular patterns. Under control conditions (25 °C), all populations showed similarly high germination (90-95%), indicating comparable baseline viability. Heat stress, however, caused a strong and population-dependent reduction in germination to 10% (H1, Wadi El-Rayan), 20% (H2, Kom Oshim) and 35% (H3, Al Kharj). SDS-PAGE revealed conserved protein bands (~ 70 and ~ 40 kDa) uniquely induced in heat-stressed seedlings from Al Kharj, suggesting population-specific thermotolerance. Total soluble protein content declined under stress in Wadi El-Rayan and Kom Oshim but partially recovered in Al Kharj, indicating differential resilience. Superoxide dismutase (SOD) activity increased consistently in all heat-stressed seedlings, highlighting a conserved antioxidant defense mechanism. LC-MS/MS analysis, based on NSAF normalization, revealed shifts in stress-related proteins between treatments and populations. Differentially expressed proteins (DEPs) were defined using thresholds of |log2 fold change| ≥ 1 and adjusted P (FDR) ≤ 0.05. Hierarchical clustering suggested that Al Kharj seedlings exhibited the most extensive proteomic adjustment at 45 °C. Correlation analyses yielded very high coefficients (|r| ≈ 0.99), but these should be interpreted cautiously due to the small number of biological replicates (n = 3). We also note that protein identification was performed against a small UniProt Zygophyllum database (868 entries), which may limit coverage and increase the risk of false positives. Overall, the data indicate both conserved (e.g., SOD induction) and population-specific responses, with the Al Kharj population showing relatively higher germination and stronger proteomic remodeling under heat stress.