Unraveling the resilience of Salicornia~europaea: physiological and molecular responses to combined environmental stresses

Plants are sessile organisms that must cope with fluctuating and often extreme environ- mental conditions through adaptive physiological and molecular mechanisms. Climate change is increasing the frequency of abiotic stress events, such as flooding (hypoxia) and salinity, which often occur simultaneously in natural habitats. Understanding how plants respond to combinations of stressors is crucial for unraveling the mechanisms underlying stress tolerance. Most previous studies have focused on stress-sensitive model plants like Arabidopsis thaliana, which mainly reveal damage responses, whereas naturally adapted halophytes offer insights into survival strategies. Salicornia europaea, a succulent halophyte of European salt marshes, exhibits high tolerance to salinity and periodic flooding. In this study, I used S. europaea to investigate the physiological, molecular, and transcriptional responses to salt, hypoxia, and simultaneous hypoxia-salt stress, to establish it as a model for simultaneous stress studies. Plants exposed to high salt concentrations (up to 2.5 M) or prolonged submergence (6 weeks) confirmed the high resilience of S. europaea under these stresses. Additionally, quantitative polymerase chain reaction (PCR) analysis revealed that hypoxia-responsive genes were induced under low oxygen conditions, but not during natural tidal flooding, suggesting efficient adaptation. Sequential exposure to salt and hypoxia altered the expression of several hypoxia-responsive genes, indicating potential crosstalk between stress response path- ways and highlighting the suitability of S. europaea as a model system for studying adaptive mechanisms under combined stresses. To investigate regulatory mechanisms, I focused on the group VII ethylene-responsive transcription factors (ERFVIIs), which are substrates of the oxygen-dependent PRT6-mediated N-degron pathway integrating salinity and hypoxia responses in A. thaliana. I assessed the involvement of ERFVIIs under simultaneous stress by exposing Arabidopsis thaliana mutants to salt and anoxia treatments. ERFVII-deficient mutants displayed better survival compared to plants lacking only HREs (Hypoxia responsive ERFVII), suggesting a specific role in adaptation. Gene expression analysis via RT-qPCR revealed differential regulation of RAPs (Related to APETALA2) and HREs under simultaneous hypoxia-salt treatment. In comparison, S. europaea displayed stronger activation of ERFVIIs as well as hypoxia- and salt-related genes, pointing to species-specific regulatory strategies. Finally, transcriptome-wide analysis using RNA sequencing revealed that combined hypoxia-salt stress induces complex transcriptional reprogramming in S. europaea. Compared to single stresses, simultaneous stress triggered about 10% more differentially expressed genes (DEGs), with up to 16% of these DEGs being unique to the combined condition. A comparative analysis of both groups was conducted, using the proportion of synergistically and antagonistically regulated genes as an indicator of their contribution to the simultaneous stress response. Using this approach, processes such as SUS-mediated sucrose degradation (Sucrose synthase) as well as proline and GABA (Gamma-Aminobutyric Acid) synthesis were found to be specifically regulated, highlighting the interplay between metabolic and regulatory pathways in plant adaptation to multiple stressors. Taken together, this work demonstrates that S. europaea represents a valuable model for investigating adaptive mechanisms to simultaneous hypoxia-salt stress. It highlights the importance of transcriptional reprogramming and ERFVII-mediated signal transduction in the integration of both stress responses. Overall, this study provides a solid foundation for future research on combined hypoxia-salt stress, by identifying a suitable model organism, delivering first insights into the molecular connections between the two stress factors, and presenting simultaneous stress response pathways for further in-depth analyses.