Poster: Intracellular Signaling
Abs #
845: Investigation into the role of calcium dependent protein kinases in pollen tubes growth in Petunia inflata
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Presenter: |
Yoon, Gyeong-Mee , gmyoon@mail.wsu.edu |
Authors | Yoon, Gyeong-Mee (A) McCubbin, Andrew G (A) | | Affiliations: |
(A): Washington State University
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After germination, pollen tubes grow through female tissues by ‘tip growth’, it is well established that Ca2+ plays a key role as a 2o messenger in this process and Calcium Dependent Protein Kinase (CDPK) activity has been implicated in this signaling pathway. Using 3’ RACE and subsequent cDNA library screening we have identified full length cDNA’s of two CDPK isoforms from Petunia inflata that are pollen specific and are expressed late in pollen development. These are the predominant and most likely the only CDPK isoforms expressed in mature pollen. We hypothesize that these two CDPK isoforms (PiPCDPK1 and PiPCDPK2 ) are key components in calcium mediated signaling in pollen tube growth. Analysis of the roles of these genes in pollen tube growth is complicated by their expression and potential roles in pollen development, as no pollen tube specific promoter is available. To circumvent these problems we have employed a biolistic system to transiently transform mature pollen with constructs driven by the LAT52 promoter. The sub-cellular localization of PiPCDPK1 and PiPCDPK2 during pollen tube growth has been investigated using GFP fusion proteins and we have performed a phenotypic analysis of pollen tubes transformed with catalytically modified forms of PiPCDPK1 and PiPCDPK2 to investigate enzyme function. Two types of construct have been used; “negative-dominance” constructs in which kinase activity has been deleted by point mutation of the catalytic site, and “unregulated” constructs, in which calcium dependency has been removed by deletion of the auto-inhibitor and calmodulin-like domains. In addition we are investigating the effectiveness of a variety of modified constructs to identify substrates of these enzymes using the yeast 2-hybrid system.
