Characterization of the Helicase activity of Pif-like Helicases from Arabidopsis thaliana and role in gene regulation and abiotic stress response

Abstract

Regulation in gene expression is rendered by the dynamic structural transitions between classical B-form DNA and non-canonical DNA folds such as G-Quadruplexes (GQS). Several proteins interact with GQSes and regulate a range of biological processes including regulation of DNA replication, transcription, and translation. Several GQS helicases resolve these structures and contribute to maintaining genomic stability. Arabidopsis has 3 homologs of Pif-Like-Helicases (AtPLHs) which have been characterized in this study. Cellular localization and tissue-specific profiling of these homologs revealed nuclear localization and expression in seedling, leaves, and flower tissues suggesting they might play a role in gene regulation and plant development. Molecular characterization of recombinant AtPLH2 and AtPLH3 suggested DNA binding, ATPase, and helicase activity of these two proteins. Further, phenotyping of Atplh mutants revealed alterated seedling root and hypocotyl growth and pollen germination. Transcriptomic profiling of mutants revealed the upregulation of genes associated with processes like systemic acquired resistance, glutathione metabolic process, cellular oxidant detoxification, β-glucosidase activity, SAM-dependent methyltransferase activity, heme binding, calmodulin binding, etc, with these genes harboring WRKY and AP2/EREB TF binding sites in their promoters. These mutants showed susceptibility to different abiotic stresses like salinity and ABA stress as well as to hydroxyurea. Further, AtPLH2 and AtPLH3 mutants showed no root elongation under low pH suggesting root elongation is affected in the absence of these genes. Our study highlights the role of these GQS helicases in regulating genes involved in root development and stress tolerance.