Investigating the Functional Role of Uncharacterized Genes in Arabidopsis.

Understanding the function of genes and the proteins they encode is essential to unravelling the mechanisms that facilitate plant adaptation to environmental stress. Many plant genes remain uncharacterized, particularly those involved in abiotic stress signaling and response. To address this, we employed a comparative approach using Arabidopsis thaliana (a model plant), Sisymbrium irio (a closely related non-extremophyte), and two halophyte species, Schrenkiella parvula and Eutrema salsugineum, which thrive in high-salinity environments. We identified two evolutionarily conserved but functionally uncharacterized Brassicaceae genes that showed stress-induced transcriptional upregulation in extremophytes: AT3G60520 (ZUP1) and AT1G02070 (ZUP2). We characterized these genes through transcriptomic analysis, protein structure prediction, reporter line imaging, and phenotypic analysis of loss-of-function and overexpression lines under abscisic acid (ABA) treatment. ZUP1/2 exhibited expression in the lateral root cap and the epidermis, tissues associated with stress signaling, and their proteins contained predicted intrinsically disordered regions (IDRs). While T-DNA mutants of both genes showed reduced primary root length under control conditions, neither loss-of-function nor overexpression lines displayed significant differences in ABA-induced root growth responses. Our findings suggest that ZUP1/2 may contribute to basal root development, but do not play a significant role in ABA-mediated root stress responses under the tested conditions. Their functions may be subtle, context-dependent, or redundant, requiring additional combinatorial or long-term stress assays to resolve. This work highlights the value of comparative genomics in unravelling gene function and attempts to inform future efforts to better understand the mechanisms of plant stress resilience.