Nuclear localization of HD-Zip IV transcription factor GLABRA2 is driven by Importin α.

GLABRA2 (GL2), a class IV homeodomain leucine-zipper (HD-Zip IV) transcription factor (TF) from Arabidopsis , is a developmental regulator of specialized cell types in the epidermis. GL2 contains a putative monopartite nuclear localization sequence (NLS) partially overlapping with its homeodomain (HD). We demonstrate that NLS deletion or alanine substitution of its basic residues (KRKRKK) affects nuclear localization and results in a loss-of-function phenotype. Fusion of the predicted NLS (GTNKRKRKKYHRH) to the fluorescent protein EYFP is sufficient for its nuclear localization in roots and trichomes. The functional NLS is evolutionarily conserved in a distinct subset of HD-Zip IV members including PROTODERMAL FACTOR2 (PDF2). Despite partial overlap of the NLS with the HD, genetic dissection of the NLS from PDF2 indicates that nuclear localization and DNA binding are separable functions. Affinity purification of GL2 from plant tissues followed by mass spectrometry-based proteomics identified Importin α (IMPα) isoforms as potential GL2 interactors. NLS structural prediction and molecular docking studies with IMPα-3 revealed major interacting residues. Split-ubiquitin cytosolic yeast two-hybrid assays suggest interaction between GL2 and four IMPα isoforms from Arabidopsis. Direct interactions were verified in vitro by co-immunoprecipitation with recombinant proteins. IMPα triple mutants ( impα- 1,2,3 ) exhibit defects in EYFP:GL2 nuclear localization in trichomes but not in roots, consistent with tissue-specific and redundant functions of IMPα isoforms in Arabidopsis . Taken together, our findings provide mechanistic evidence for IMPα-dependent nuclear localization of GL2 and other HD-Zip IV TFs in plants. One sentence summary: GLABRA2, a representative HD-Zip IV transcription factor from Arabidopsis , contains an evolutionarily conserved monopartite nuclear localization sequence that is recognized by Importin α for translocation to the nucleus, a process that is necessary for cell-type differentiation of the epidermis.

The START domain mediates Arabidopsis GLABRA2 dimerization and turnover independently of homeodomain DNA binding.

Class IV homeodomain leucine-zipper transcription factors (HD-Zip IV TFs) are key regulators of epidermal differentiation that are characterized by a DNA-binding HD in conjunction with a lipid-binding domain termed steroidogenic acute regulatory-related lipid transfer (START). Previous work established that the START domain of GLABRA2 (GL2), a HD-Zip IV member from Arabidopsis (Arabidopsis thaliana), is required for TF activity. Here, we addressed the functions and possible interactions of START and the HD in DNA binding, dimerization, and protein turnover. Deletion analysis of the HD and missense mutations of a conserved lysine (K146) resulted in phenotypic defects in leaf trichomes, root hairs, and seed mucilage, similar to those observed for START domain mutants, despite nuclear localization of the respective proteins. In vitro and in vivo experiments demonstrated that while HD mutations impair binding to target DNA, the START domain is dispensable for DNA binding. Vice versa, protein interaction assays revealed impaired GL2 dimerization for multiple alleles of START mutants, but not HD mutants. Using in vivo cycloheximide chase experiments, we provided evidence for the role of START, but not HD, in maintaining protein stability. This work advances our mechanistic understanding of HD-Zip TFs as multidomain regulators of epidermal development in plants.

Making glue from seeds and gums: Working with plant-based polymers to introduce students to plant biochemistry.

Plants and plant products are key to the survival of life on earth. Despite this fact, the significance of plant biochemistry is often underrepresented in science curricula. We designed an innovative laboratory activity to engage students in learning about the biochemical properties of natural polymers produced by plants. The focus of the hands-on activity is on mucilages and gums, which contain complex polysaccharides that have applications in industry. The 1.5-h activity is organized into three laboratory exercises. It begins with a demonstration of the water absorption property of seed coat mucilage upon hydration of seeds from psyllium, a plant that is grown commercially for mucilage production. The second exercise involves microscopy of a variety of plant seeds stained with ruthenium red dye to visualize pectin polysaccharides of the seed mucilage. Students learn about phenotypic variation among plant species and how the seed coat mucilage is beneficial to keep seeds hydrated during germination. The third exercise highlights an industrial application of plant gums as adhesives. The students prepare edible glue made with gum arabic, a type of plant polymer from the dried exudate of the Acacia plant. This three-part activity has been implemented in conjunction with a Girls Researching Our World (GROW) summer workshop for sixth to eighth graders over a 4-year period. It may be adapted as a laboratory activity for students of all ages, for example, to enhance biochemistry education for high-school students or undergraduate non-majors. © 2019 International Union of Biochemistry and Molecular Biology, 47(4):468-475, 2019.