Investigating the Web of Calcium-Dependent Protein Kinase Mediated Signaling Pathways in the Polar Growth of Pollen Tubes

Nolan Scheible

doi:10.7273/000006363

Calcium is an important second messenger in plant cell signaling pathways. Calcium-mediated signaling is critical to many developmental events in the plant life cycle, including the regulation of pollen tube growth. Pollen plays a pivotal role in plant reproduction by producing and transporting sperm cells to be delivered to the egg and facilitate fertilization. Not only is pollen tube growth a prerequisite for the plant life cycle to proceed, but the economic relevance of this process has major implications to food security. The crops we consume as fruits or seeds are the direct result of succesfull pollen tube growth and thus the efficiency of this process has humanitarian ramifications beyond that of the plant life cycle. There are many signaling pathways that overlap with one another to coordinate pollen tube growth. Polar growth, where growth is restricted to a single expansion point, requires the coordination of cell processes that provide the accurate deposition and recycling of new cell wall materials. Among these processes the role of steep calcium pulses at the tube apex followed by expansion of the tube tip has been demonstrated. Increased calcium levels in the tube tip result in the activation of different down-stream calcium-activated proteins. Our lab previously identified a calcium-depdendent protein kinase (CDPK), CDPK1, as one of these proteins activated by calcium. When CDPK1 is over-expressed in pollen the tube tip becomes swollen and spherical, supporting a role for this protein in the polar growth of pollen tubes. Here substrates of CDPK1 that are involed in the polar growth of pollen tubes have been identified. The first protein investigated in this work is a rho guanine dissociation inhibitor (RhoGDI1) that was identified as interacting with CDPK1 using a yeast 2-hybrid system. Using stable transgenic tobacco it was shown that co-expression of RhoGDI1 partially rescued the CDPK1 over-expression phenotype in pollen tubes. This result supports a genuine interaction between these two proteins. As this was the only candidate interaction identified using the yeast 2 hybrid system alternative experiments were designed to in an effort to identify additional CDPK1substrates. An pull-down experiment intended to isolate CDPK1 and any interacting protein partners from pollen lysate of stable transgenic tobacco was performed. This resulted in identification of 123 of potential CDPK1 substrates. Two were selected for further characterization, a 14-3-3 protein called growth factor 14 (GF14) and a rho GTPase activating protein (RhoGAP) called rho GTPase enhancer 1 (REN1). Studies were carried out in stable transgenic tobacco to confirm or deny the interactions of these proteins with CDPK1. These studies are imperative for understanding the role calcium plays in vital processes such as pollen tube growth. Calcium signaling is likely to overlap and intersect with many other signaling pathways and hence a holistic view of the processes governed by calcium is paramount if we ever hope to manipulate those processes.

Investigating the Web of Calcium-Dependent Protein Kinase Mediated Signaling Pathways in the Polar Growth of Pollen Tubes

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