Deciphering the role of Pif Helicases during abiotic stress and reproductive development in Arabidopsis thaliana

Abstract

Regulation in gene expression is rendered by the dynamic structural transitions between newlineclassical B-form DNA and non-canonical DNA folds such as G-Quadruplexes (GQS). newlineSeveral proteins interact with GQSes and regulate a range of biological processes including newlineregulation of DNA replication, transcription, and translation. Several GQS helicases newlineresolve these structures and contribute to maintaining genomic stability. Arabidopsis has 3 newlinehomologs of Pif-Like helicases (AtPLH-AtPLH1, AtPLH2, and AtPLH3), which have been newlinecharacterized in this study. Cellular localization and tissue-specific profiling of these newlinehomologs revealed nuclear localization and expression in seedling, leaves, and flower newlinetissues suggesting they might play a role in gene regulation and plant development. newlineMolecular characterization of recombinant AtPLH2 and AtPLH3 suggested DNA binding, newlineATPase, and helicase activity of these two proteins, and site-directed mutagenesis in the newlineWALKER domain revealed the perturbation in the ATPase activity supports the newlineconservation of active site residues between prokaryotes and eukaryotes. Further, double newlinemutants were generated and phenotyping of single and double mutants of AtPLHs revealed newlinemorphological alterations in the seedling root, hypocotyl growth, and pollen tube newlinemorphology. Transcriptomic profiling of mutants revealed the upregulation of genes newlineassociated with processes like systemic acquired resistance, glutathione metabolic process, newlinecellular oxidant detoxification, and#946;-glucosidase activity, SAM-dependent methyltransferase newlineactivity, heme binding, calmodulin binding, etc, with these genes harboring WRKY and newlineAP2/EREB TF binding sites in their promoters. The expression analysis of AtPLH newlinehomologs under various abiotic stresses like ABA, NaCl, and HU suggested regulation of newlineAtPLH gene expression under these conditions, and mutants showed susceptibility to newlinedifferent abiotic stresses like salinity and ABA stress and hydroxyurea. Further, AtPLH2 newlineand AtPLH3 mutants showed no root elongation under low pH suggesting root.