Cyclic Nucleotide Gated Channels 7 and 8 Are Essential for Male Reproductive Fertility |
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Authors: | Meral Tunc-Ozdemir Claudia Rato Elizabeth Brown Stephanie Rogers Amanda Mooneyham Sabine Frietsch Candace T Myers Lisbeth Rosager Poulsen Rui Malhó Jeffrey F Harper |
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Institution: | 1. Department of Biochemistry, University of Nevada, Reno, Nevada, United States of America.; 2. Universidade de Lisboa, Faculdade de Ciências de Lisboa, BioFIG, Lisboa, Portugal.; 3. Department of Plant Biology and Biotechnology, Centre for Membrane Pumps in Cells and Disease (PUMPKIN), University of Copenhagen, Danish National Research Foundation, Frederiksberg, Denmark.; National Taiwan University, Taiwan, |
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Abstract: | The Arabidopsis thaliana genome contains 20 CNGCs, which are proposed to encode cyclic nucleotide gated, non-selective, Ca2+-permeable ion channels. CNGC7 and CNGC8 are the two most similar with 74% protein sequence identity, and both genes are preferentially expressed in pollen. Two independent loss-of-function T-DNA insertions were identified for both genes and used to generate plant lines in which only one of the two alleles was segregating (e.g., cngc7-1+/−/cngc8-2−/− and cngc7-3−/−/cngc8-1+/−). While normal pollen transmission was observed for single gene mutations, pollen harboring mutations in both cngc7 and 8 were found to be male sterile (transmission efficiency reduced by more than 3000-fold). Pollen grains harboring T-DNA disruptions of both cngc7 and 8 displayed a high frequency of bursting when germinated in vitro. The male sterile defect could be rescued through pollen expression of a CNGC7 or 8 transgene including a CNGC7 with an N-terminal GFP-tag. However, rescue efficiencies were reduced ∼10-fold when the CNGC7 or 8 included an F to W substitution (F589W and F624W, respectively) at the junction between the putative cyclic nucleotide binding-site and the calmodulin binding-site, identifying this junction as important for proper functioning of a plant CNGC. Using confocal microscopy, GFP-CNGC7 was found to preferentially localize to the plasma membrane at the flanks of the growing tip. Together these results indicate that CNGC7 and 8 are at least partially redundant and provide an essential function at the initiation of pollen tube tip growth. |
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