Graduate Student Kansas State University Manhattan, Kansas
Body of Abstract: GLABRA2 (GL2), a class IV homeodomain leucine-zipper (HD-Zip IV) transcription factor from Arabidopsis, plays a critical role in the differentiation of trichomes as well as the epidermis of the root and seed. GL2 contains a putative monopartite nuclear localization sequence (NLS) partially overlapping with its homeodomain. We show that NLS deletion or alanine substitution of basic residues (KRKRK) affects nuclear localization and results in a gl2 null mutant phenotype. Fusion of the predicted 13 amino acid NLS (GTNKRKRKKYHRH) to the enhanced yellow fluorescent protein (EYFP) is sufficient for its nuclear localization in roots. The functional NLS is conserved in a distinct subset of HD-Zip IV members that includes PROTODERMAL FACTOR2 (PDF2). GL2 immunoisolation from plant tissues in tandem with mass spectrometry-based proteomics identified several importin alpha isoforms as potential interactors. NLS structural prediction and molecular docking studies with importin alpha revealed major interacting residues, including those that were targeted for alanine substitution. Confocal imaging and electrophoretic mobility shift experiments with PDF2 indicate that DNA binding and nuclear localization are separable functions. Split-ubiquitin-based cytosolic yeast two-hybrid assays showed interaction between GL2 and five importin alpha isoforms from Arabidopsis. The interactions were further verified by co-immunoprecipitation using a Nicotiana benthamiana transient expression system. Importin alpha triple mutants (imp-a1,2,3) show defects in EYFP:GL2 nuclear localization in trichomes but not in roots, consistent with tissue-specific functions of importin alpha isoforms in Arabidopsis. Taken together, our findings provide mechanistic evidence for importin-alpha dependent nuclear localization of HD-Zip IV transcription factors in plants.