The long-term goal of this project is to understand the functions of calcium-dependent protein kinases (CPKs) in regulating many aspects of plant growth and development. CPKs (which are defined by a novel structure in which the kinase domain is fused to a calmodulin-like regulatory domain) appear to be unique to plants and certain single-celled organisms, and have not been found in animals. In the model plant Arabidopsis, there are 34 CPKs, 12 of which are expressed in pollen. While many plant CPKs are thought to be involved in regulating the response to environmental stresses, such as drought, heat and cold, recent genetic evidence has been obtained that identifies two of these CPKs as critical to pollen tube tip growth, which is a developmental program. Genetic knockout of CPK17 and 34 results 1) in pollen tubes that are short and slow growing and that are disrupted in their ability to locate and fertilize ovules, and 2) in plants that are essentially sterile. The ability to restore normal fertility by transferring CPK34 genes into these plants provides an unprecedented opportunity to determine the features of a CPK that are important for their functions.
A key hypothesis guiding the research is that specific functions of CPK17 and 34 are encoded in the kinase domain. The aims of this project include 1) defining the molecular pathways by which these CPKs promote pollen tube tip growth and tropism, 2) delineating the cellular processes regulated by these CPKs, starting with endocytosis and exocytosis at the tip of a growing pollen tube, and 3) identifying how these CPKs regulate vesicle trafficking and cytoskeletal dynamics, which are critical processes in polarized pollen tip growth. This research will provide new insights into how calcium signals are coded and decoded in plant systems.
Broader impacts. The research will have additional impacts that derive from educational and mentoring contributions. The Harper lab is actively engaged in mentoring research activities at all academic levels, from early career assistant professors to undergraduates. The planned research will provide training for both undergraduate and graduate student in the area of molecular biology, confocal microscopy, genetics and proteomics. Project results will be communicated to the public through publications, data base contributions, scientific seminars, and lectures to undergraduates and K-12 students.
The long-term goal of the research is to understand a fundamental aspect of sexual reproduction in flowering plants – how pollen tubes grow, locate ovules, and discharge sperm cells for double fertilization of eggs and central cells. In a model plant Arabidopsis thaliana, the loss of two redundant calcium-dependent protein kinases (CPKs 17 and 34) results in pollen tubes that are short, slow growing, and dysfunctional in their ability to locate ovules. These pollen deficiencies result in plants with a near-sterile phenotype. This phenotype provided an opportunity to screen for additional mutations that would suppress the infertility and allow the cpk17/34 mutant pollen to grow. This experimental strategy identified a "suppressor gene" that provides an unexpected connection between the calcium-signaling pathway mediated by CPK17/34 and the function of a specific protein thought to be involved in modifying cell wall polysaccharides. For plant cells that use turgor pressure to drive cell expansion, the structure and rigidity of the cell wall needs to be carefully regulated to allow the cell to grow without a catastrophic cellular rupture. The insights and tools developed in this research provide a unique opportunity to gain basic knowledge about how transient changes in calcium concentrations are decoded into signals that regulate cell wall biogenesis and other processes important for cell growth. This research has also provided evidence that modifications to pollen cell wall polysaccharides are important for how pollen cope with the stress of hot and cold temperatures. The ability of pollen to cope with temperature stresses is already a major agronomic problem, which might become critical in the context of climate change. The goal of this research is to obtain fundamental scientific insights into questions about cell growth and plant reproduction that will enable future research into making crop plants more productive, especially in the context of improved tolerance to biotic and abiotic stress.
