Graduate Research Assistant The University of North Carolina at Chapel Hill
All plant biomass originates from populations of self-renewing stem cells located in specialized regions called meristems. Control of meristem size is critical in development and is inexorably linked to crop yield. In Arabidopsis, meristem size is controlled by the CLAVATA signaling pathway. The signaling output of this pathway modulates meristem size by limiting stem cell proliferation and allowing for differentiation to occur. Many of the signaling components have been discovered; however, little is known about how they are regulated at the molecular level. Two redundant class 2C protein phosphatases, POLTERGEIST (POL) and POLTERGEIST-LIKE 1 (PLL1), are known to negatively regulate CLAVATA signaling, but how they do so is unknown. Mutation in one of these genes causes no noticeable phenotype, but the double mutant is seedling lethal, indicating its importance in development. In order to elucidate how POL regulates meristem size, we performed an in-depth analysis of POLTERGEIST function using bioinformatics, genetics and biochemistry. We identified multiple novel, conserved, and family-specific domains which are enriched in serine residues. Many of these residues were determined to be phosphorylated, predicted to be phosphorylated, or a combination of the two. This suggested that POL is regulated, post-translationally, via phosphorylation. To determine if phosphorylation regulates POL function, we generated phospho-site substitution variants of POL, either phospho-mute (Ser >Ala) or phospho-mimetic (Ser >Asp), and tested their ability to complement the seedling lethal mutant phenotype. We found that the phospho-mute versions of POL complemented the phenotype while the phospho-mimetic did not. Based on our evidence, we postulate that POL function is inactivated via phosphorylation of two novel domains found specifically in POL family members. This information furthers our understanding of the molecular pathways that regulate stem cell development and provides enticing targets for modification in crop species to increase yield.
Coauthors: Zack Nimchuk, Dr. – The University of North Carolina at Chapel Hill