Molecular Modulation of the WRKY53, WRKY18, and WRKY25 Regulatory Subnetwork of Leaf Senescence

Abstract

Senescence represents the final stage of plant development and strongly impacts yield quantity and quality. This regulated process involves epigenetic, transcriptional, and biochemical mechanisms and is influenced by phytohormones, biotic and abiotic stresses, signaling molecules such as Ca²⁺ and H₂O₂, and intrinsic factors like developmental stage and nutrient status. In this doctoral research, I investigated the regulatory network governing senescence, focusing on transcription factors of the WRKY family. Among them, WRKY53 is a central regulator that activates senescence-associated genes (SAGs) and integrates environmental signals, particularly through H₂O₂-dependent activation. WRKY transcription factors form a network, since their promoters often contain WRKY binding motifs. Within this network, WRKY25 activates and WRKY18 represses WRKY53, yet mutants of either gene show accelerated senescence, indicating complex regulation. This work examined the WRKY25–WRKY18–WRKY53 subnetwork in detail, revealing that WRKY25 functions as a dual regulator, sensing reactive oxygen species (ROS) and balancing the network under oxidative stress. Domain-specific analyses highlighted distinct roles for the N- and C-terminal regions of WRKY25. Based on these findings, WRKY25 is proposed to act as a redox switch modulating WRKY53 expression through other network components. A novel feedback mechanism was also identified between H₂O₂, catalases, and WRKY53. WRKY53 is transcriptionally induced by H₂O₂, while catalases interact directly with WRKY53 at the protein level, resulting in mutual inactivation. This illustrates a fine-tuned redox-dependent loop. Beyond WRKYs, WRKY53 is controlled by other transcription factor families. Among them, the HD-ZIPIII factor REVOLUTA (REV) was characterized as a positive regulator of WRKY53 and its H₂O₂ response. Regulation of REV activity by TIFY proteins was studied in depth, suggesting two mechanisms: a jasmonate (JA)-independent pathway through TIFY8 controlling REV in senescence, and a JA-dependent pathway involving PEAPODs and several JAZ proteins. Altogether, this study uncovers new mechanistic insights into transcriptional and post-transcriptional regulation of plant senescence. It highlights the ability of transcription factors to perceive environmental signals and integrate them into developmental programs. These findings deepen our understanding of senescence and open promising avenues for crop improvement by manipulating senescence pathways to optimize yield and quality. untranslated