1. Genetic evidence for essential calcium transporters in pollen growth and fertilization. Sabine Frietsch 1,3, Shawn M. Romanowsky 1,2, Morton Schiøtt 4, Michael G. Palmgren 4, Jeffrey F. Harper 1,2 1 The Scripps Research Institute, Cell Biology, 10550 N. Torrey Pines Rd, 92037 La Jolla, CA, USA 2 University of Nevada, Biochemistry, 1664 North Virginia Str, 89557 Reno, CA, USA 3 University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0116 4 The Royal Veterinary and Agricultural University, Plant Biology, Thorvaldsensvej 40, DK-1871 Frederiksberg, Denmark sfrietsch@uscd.edusfrietsch@uscd.edu and jfharper@unr.edujfharper@unr.edu Introduction: Calcium dynamics are thought to play a central role in pollen development, as evidenced by pharmaco-chemical approaches and visualization of calcium gradients and oscillations. Using Arabidopsis as a model system, we provide the first genetic evidence to support a model in which calcium signals are natural regulators of pollen tube growth and fertilization. Calcium signals are largely controlled by influx (through channels) and efflux (through pumps and antiporters). We have identified T-DNA gene disruptions in all 14 calcium pumps, and all 20 cyclic nucleotide gated channels (CNGCs). From this set of mutants, distinct pollen specific phenotypes have been found for disruptions of ACA9 (a calmodulin activated plasma membrane calcium pump) and CNGC18 (a cyclic nucleotide and calmodulin regulated putative calcium channel). Calcium Pump Mutation aca9 Reduces Pollen Tube Growth Potential (in vivo) cngc18 -/+ have full seed set indicating that the defect is very different than the aca9 phenotype. Reciprocal crosses and out-crossing to the male sterile mutant, ms-1 show that cngc18 is male sterile. CONCLUSIONS: funiculus aca9 disruption results in partial male sterility WTaca9 Longest pollen tube Aniline blue stain WT ACA9promoter::GUS ACA9 appears to function as a plasma membrane pump in pollen aca9 disruption results in >50% reduced frequency of synergid penetration (discharge) required for fertilization aca9 -/- blocked fertilized Synergid penetration penetration / discharge Confocal image of GFP merged with DIC image No synergid pentration or sperm release Discharge + Discharge - Discharge +: Wild type 96% (n=178) aca9 41% (n=247) CNGC18 disruption results in complete male sterility No homozygous cngc18 in over 400 F1 progeny of two independent gene disruption lines. Homozygous gene disruption lines in all CNGCs but CNGC18. Alexander staining for pollen viability in the quartet background (qrt) shows that cngc18 pollen is normal developed and viable. Expression profile of CNGCs during pollen development Modified after Maeser et al., 2001 CNGC8 and CNGC18 are highly expressed in mature pollen. Only disruption of CNGC18 results in male sterility. 100x cngc18 pollen tubes germinate, but only grow a short distance, with a “kinky-like” non-directional growth, often prematurely terminating with a bursting event. cngc18 pollen germinate but has impaired tube growth in vitro 400x control, qrt (-/-)cngc18 (-/+), qrt (-/-) In vitro germination assays in the quartet background (pollen doesn’t separate during development). control, qrt (-/-) cngc18 (-/+), qrt (-/-) Working model for a CaM regulated calcium oscillator aca9 is the first mutant with a defect in pollen/ovule interaction. CNGC18 is the only CNGC essential for the plant life cycle. CNGC18 is the first potential calcium channel that was found to be involved in pollen tube growth. ACA9 and CNGC18 could be involve in a CaM regulated calcium oscillator, that is essential for pollen tube growth and fertilization. aca9 -/- plants display reduced pollen tube growth and >50% reduction of synergid penetration (discharge). ACA9: CNGC18: Model: cngc18 pollen germinates with underdeveloped, kinky pollen tubes, that often burst at the tip. Disruption of ACA9 results in partial male sterile plants with shorter siliques and reduced seed set. Disruption of CNGC18 results in complete male sterility. Honys and Twell, 2003, Extracted from NASC database WT,col cngc18 (-/+)