054 | Biostimulant-mediated mitigation of hypoxic stress and recovery in lettuce under flooding conditions

Chiara Dellapiana¹, Chiara Agliassa², Francesco Caldo¹, Moez Maghrebi¹, Valeria Contartese², Giuseppe Mannino¹, Gianpiero Vigani¹ | ¹Department of Life Sciences and Systems Biology, University of Turin, Italy; ²Green Has Italia SpA, Canale [AT], Italy.

Climate change has intensified the alternation between drought and heavy rainfall, making flooding a major global constraint to crop productivity through reduced oxygen availability. As obligate aerobic organisms, plants require oxygen and its by-products to perform essential metabolic functions, growth, development and acclimation responses to changing environmental conditions. Within this scenario, the development and validation of innovative, sustainable biotechnological tools, such as plant biostimulants, are gaining attention as promising strategies to enhance crop resilience to abiotic stresses. Acquiring knowledge of biostimulant mode of action at physiological, cellular and molecular levels is therefore crucial to define the efficiency of these agronomic strategies. This project aimed to investigate the effects of a new biostimulant prototype on the responses of lettuce to oxygen deprivation (hypoxia) under waterlogging involving root system submersion and complete plant submergence. Plant responses to flooding stress were evaluated through: (i) measurement of photosynthetic efficiency parameters (Fv/Fm, PIabs, ABS/RC and ET0/CSm), (ii) assessment of leaf oxidative stress by spectrophotometric assays targeting stress markers (MDA and H₂O₂) and enzymatic (SOD and POX) and non-enzymatic antioxidant machinery (proline), (iii) evaluation of plant nutritional status via quantification of inorganic anions at the leaf level by capillary electrophoresis (complete submergence only), and (iv) analysis of hypoxia marker gene expression (LsADH1, LsADH2, LsPCO1, LsHRE1, LsHb2, LsSAD6, LsLBD41 and LsERF071; complete submergence only). Results confirmed the establishment of a stress condition following flooding, as indicated by an overall impairment of photosynthetic efficiency, increased oxidative stress markers coupled with modulation of enzymatic scavengers and non-enzymatic antioxidants, a worsening of plant nutritional status based on leaf anion content, and overexpression of selected hypoxia-responsive genes. In contrast, biostimulant treatment induced stress-type-specific responses in lettuce subjected to flooding, suggesting a modulation of plant acclimation mechanisms rather than a generic stress attenuation. While photosynthetic efficiency decreased under both waterlogging and complete submergence, biostimulant-treated plants exhibited an enhanced recovery of photosynthetic parameters. Biochemical analyses revealed mitigation of oxidative stress in both conditions, with higher proline levels and lower MDA during recovery from waterlogging, and lower MDA and H₂O₂ levels associated with enhanced antioxidant enzyme activities (SOD and POX) under complete submergence. Improved nutritional status and reduced transcriptional induction of hypoxia-responsive genes in submerged plants further supported the role of the biostimulant in alleviating hypoxic stress, likely by lowering stress perception and limiting hypoxia-related molecular responses, thereby promoting a more efficient post-stress recovery. Overall, these findings demonstrate that the tested biostimulant prototype contribute in mitigating hypoxia-induced stress in lettuce. This work highlights the potential of biostimulants as innovative biotechnological tools for improving crop resilience, supporting their development for sustainable agriculture under climate-driven environmental challenges.