Investigation of the Role of Kinase-Associated Protein Phosphatase (KAPP) in Pistil-Pollen Interactions in Brassica napus (canola)
Date
2021-06-25
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Abstract
Climate change and our increasing population threatens the availability of sustainable food sources, creating greater pressure to find solutions. Increasing crop yield can mitigate these problems. Given that seeds are the most important agronomic trait, a key component of this reproductive success is pollination. The pollination pathway, both compatible and self-incompatible (SI), is comprised of complex signalling mechanisms which govern pollen-pistil communications. Even with significant contributions to our understanding of the pollination response through the identification of key players and its signalling mechanism, there remains a gap in our understanding of the SI pathway.Although kinase-associated protein phosphatase (KAPP) can interact in vitro with various receptor kinases, for decades, its specific role in SI remained unknown. In this study, biochemical approaches determined that KAPP was highly expressed in mature stigmas with poor expression in pollen. While compatible pollen did not affect KAPP levels, following SI pollination, KAPP levels were significantly increased. Through transgenic approaches, overexpressing only the phosphatase type 2C (PP2C) domain of KAPP sufficiently reduced pollen acceptance, leading to shorter pods and reduced seed production. These observations indicate that KAPP could be a positive regulator in the incompatibility response where it acts through its PP2C domain.When a PP2C domain mutated in the mitogen-activated protein kinase (MAPK) docking site (KAPP?290M) was overexpressed, a more compatible phenotype relative to PP2C overexpression was observed in the KAPP?290M transgenic lines. However, biochemical analysis revealed that this phenotype was not due to the inability of the KAPP?290M to dephosphorylate MPK4 as the mutated version KAPP?290M was able to effectively dephosphorylate MPK4 in vitro. Confocal imaging of KAPP?290M-RFP expression indicated that, lack of these key MAPK docking residues resulted in localization of KAPP?290M-RFP from the cytosol to the plasma membrane. Collectively, in this study, I have identified KAPP as a positive regulator of SI acting through its PP2C domain and provided evidence for the requirement of the MAPK docking site for precise intracellular localization. Therefore, this study bridges the knowledge gap in SI and provides another protein which can be manipulated to create novel hybrids to improve crop yield.
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Ribano, A. B. (2021). Investigation of the Role of Kinase-Associated Protein Phosphatase (KAPP) in Pistil-Pollen Interactions in Brassica napus (canola) (Master's thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.