The Role of Two Group I WRKY Transcription Factors and Their Interaction Protein in Plant Development and Responses to Environmental Cues

Drought stress remains a major limitation to global crop productivity, necessitating a deeper understanding of plant stress responses for effective bioengineering strategies. WRKY transcription factors play pivotal roles in abiotic stress regulation, yet their specific contributions to drought tolerance remain insufficiently characterized. In this study, we employed a multidisciplinary approach encompassing stem cell research, genetic analysis, genome editing, transcriptomics, hyperspectral photometer imaging, artificial intelligence-backed stomata imaging, and physiological assessments using the LI-COR Photosynthesis System to investigate drought tolerance and development mechanisms. A double knockout of two WRKY genes resulted in early senescence, suggesting their roles in balancing growth and stress responses. Comparative transcriptomic analysis of wildtype and mutant plants under drought conditions revealed significant upregulation of an R3H-domain protein in the WRKY double knockout, indicating a potential downstream regulatory link. Functional characterization of R3H knockouts demonstrated increased drought sensitivity, implicating this protein as a key modulator of stress tolerance. Furthermore, one of the WRKY proteins was found to interact with an NHL (NDR1/HIN1-like) protein, a class of stress-associated proteins implicated in membrane integrity and signaling. Hyperspectral imaging and AI-powered stomatal imaging enabled detailed physiological profiling, providing novel insights into the phenotypic consequences of these genetic modifications. Comprehensive physiological assays, including photosystem II efficiency, stomatal conductance, transpiration rate, and LI-COR-based gas exchange measurements, further elucidated the distinct physiological signatures associated with these mutations. This integrative study advances our understanding of the WRKY-R3H-NHL network in drought stress regulation and plant development, and contributes valuable genetic resources for engineering drought-resilient crops to enhance food security in the face of climate change.