Designing Tools to Probe the Calcium-dependent Function of Arabidopsis Tonneau2

• Main Author: Oremade, Oladapo O.
• Other Authors: Wright, Amanda
• Format: Others
• Language: English
• Published: University of North Texas 2013
• Subjects: Cell division; PP2A phosphotase; prephosphase band
• Online Access: https://digital.library.unt.edu/ark:/67531/metadc407800/

Plants possess unique features in many aspects of development. One of these features is seen in cell wall placement during cytokinesis, which is determined by the position of the preprophase band (PPB) and the subsequent expansion of the phragmoplast that deposits the new cell wall. During phragmoplast expansion, the phragmoplast tracks to the cortical division site, which was delineated by the PPB. Thus the position of the PPB determines the orientation of the division plane. In Arabidopsis thaliana, TONNEAU2 (TON2) is required for PPB formation and has been shown to interact with a type A subunit of the PP2A phosphatase in the yeast two-hybrid system. In Arabidopsis tonneau2 (ton2) mutants, abnormalities of the cortical microtubule cytoskeleton, such as disorganization of the interphase microtubule array and lack of PPB formation before mitosis markedly affects cell shape and arrangement as well as overall plant morphology. Loss of dcd1/add1, the maize ton2 homologues gives rise to a similar phenotype in Zea mays. The TON2 protein has two EF hand domains which are calcium-binding sites. Since calcium has been known to play key roles in several areas of plant functioning, the following question was raised: “Does calcium binding contribute to the localization and function of TONNEAU at the PPB?” To address this question, a series of constructs were generated to determine if TON2 binds calcium. Additionally, Ca2+ binding sites were mutated in constructs containing the TON2 gene fused to GFP or YPF. These constructs were then transformed into ton2 mutant plants and the localization of TON2 fusion protein and whether the construct is capable of rescuing the mutant phenotype were observed. Although, localization of TON2 to the PPB was not observed, the presence of the constructs were confirmed in the transformed plants using selection markers and by observing fluorescence under a confocal microscope.