H2O2 priming: Biostimulation, drought tolerance and DNA methylation profile with intergenerational impact in tobacco plant

H₂O₂ is a promising priming agent due to its role in stress-response pathways and in the regulation of gene expression. Several studies have shown that H₂O₂ elicitation improves stress responses. However, information regarding of the intergenerational stress memory of the H₂O₂ priming is limited. Therefore, in this study, we evaluated the stress memory induced by H₂O₂ priming in tobacco plants by testing hydric stress phenotypic response and changes in DNA methylation in the parental priming line and in the next generation. Priming consisted of three foliar applications of 200 mM H₂O₂ every 5 days on 4-week-old plants. DNA methylation profiling was performed by Whole Genome Bisulphite Sequencing (WGBS). Hydric stress challenge consisted of three conditions: control (100 %), moderate (40–45 %) and severe (20–25 %) stress according to the available water field capacity. Plant response to the challenge was evaluated by morphological, biochemical, and molecular attributes. The results showed that H₂O₂ priming displayed a biostimulant effect on vegetative and root development, preventing growth stunting under drought conditions. Plant antioxidant activity was enhanced, as reflected by the decrease in endogenous H₂O₂. In drought conditions, proline content, CAT activity and gene expression of CHS, PIP1 and AQP1 genes were enhanced in the primed-plants (H), their progeny (F1), and the primed-progeny group (FH). DNA methylation profile revealed 795 differentially methylated cytosines (DmCs) in the progeny, with 89 associated with genes involved in cellular response to environmental stimuli. An interaction network highlighted stress response with flavoreductase enzyme as central interaction node, and in the second layer the Repressor of Silencing 1 (ROS1) gene. Thus, the findings suggest that H₂O₂ priming may induce intergenerational memory, presenting a potentially cost-effective strategy in stress management in agriculture.