ABS 002: Defining the Subcellular Localization of Protein Phosphatase Complexes that Regulate Leaf Expansion

Michael Medeiros ¹ , Alex Tomkinson ¹ , Alison DeLong ²

¹ Brown University, RI, USA
² Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University

Van Wickle (2025) Volume 1, ABS 002

Introduction: Protein phosphatase 2A (PP2A) is a large, highly conserved, and highly regulated enzyme family. It includes heterotrimeric protein complexes that have gained attention due to their roles in cell cycle regulation and other key processes in eukaryotes. PP2A complexes comprise three subunits: the scaffolding A subunit, variable B subunit, and catalytic C subunit containing the active site. The binding of the regulatory B subunit to an A-C core complex confers substrate specificity and forms the PP2A holoenzyme. The B subunits are encoded by several distinct gene families conserved throughout eukaryotes. Within these conserved families, the B72 gene family in Arabidopsis contains two genes that play a key role in regulating PP2A activity, B16 and B17. I will be investigating the functions of these genes, using B17 fusions to the green fluorescent protein (GFP) to analyze the subcellular localization properties of B72 subunits.
In the DeLong lab, mutants have been isolated with knockouts of the B16 and B17 genes. While single b16 or b17 mutants show normal phenotypes, b16b17 mutants show increased leaf size. This phenotype indicates that both genes are negative regulators of leaf expansion, but the mechanism linking PP2A activity to leaf expansion is unknown. We will use the GreenGate modular cloning system to investigate this unknown mechanism by creating B17 fusion constructs with a GFP tag. Once the constructs are assembled and verified in E. coli, we will introduce them into Agrobacterium. The Agrobacterium will be used to perform a floral dip transformation on A. thaliana plants. Through selection for antibiotic resistance, we will obtain and characterize several transgenic lines for each construct. We will use fluorescence microscopy and complementation assays (comparing transgenic and wild-type leaf sizes) to characterize the transgenic plant phenotypes.

Methods: We are investigating the functions of these genes, using constructs of B17 fusions to the green fluorescent protein (GFP) to analyze the functional domains and subcellular localization properties of B72 subunits. Each of the constructs aims to address how the B17 function is altered when protein is anchored in the nucleus, and if a free C-terminus/N-terminus required for complementation. Our strategy is to introduce these constructs into the double mutant background (b16b17-2) to test their ability to restore a normal leaf size phenotype, as well as using fluorescence microscopy to address the subcellular localization of each construct.

Results: We expect that the pB17IGR: B17FL control construct will fully complement the b16b17-2 mutant. GFP-only constructs should show no complementation activity. Complementation by all other constructs will be compared to these controls
If the B17 protein requires a free N- or C-terminus to function, GFP fusions at one or both ends and between the N and C-termini of the full-length protein will fail to complement.
If the B17 protein is limited by nuclear localization, we expect GFP-NLS fusions to either end of the protein to fail to complement, resulting in a large leaf phenotype.

Discussion: B72 proteins are highly conserved in eukaryotes, while their localization and activity remain uncharacterized; our experiments aim to reveal where the B72 family member B17 functions in regulating leaf size, both in its expression pattern and its subcellular localization. We have assembled nine constructs, six of which have already been transformed into Arabidopsis, and two have already produced stable, GFP-expressing transgenic lines. Moving forward, we aim to further characterize B17 subunit function and regulation.