Journal Article

Cyclic Nucleotide Gated Channels 7 and 8 Are Essential for Male Reproductive Fertility

Rato, Claudia, Rogers, Stephanie, Poulsen, Lisbeth Rosager, Harper, Jeffrey F., Tunc-Ozdemir, Meral, Mooneyham, Amanda, Brown, Elizabeth, Frietsch, Sabine, Myers, Candace T., Malhó, Rui

2013

Overview

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.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: alleles / Amino acid sequence / Arabidopsis / Arabidopsis - cytology / Arabidopsis - genetics / Arabidopsis - metabolism / Arabidopsis - physiology / Arabidopsis Proteins - genetics / Arabidopsis Proteins - metabolism / Arabidopsis thaliana / BASIC BIOLOGICAL SCIENCES / Biochemistry / Biology / Calcium binding proteins / Calcium channels / Calcium permeability / Calcium-binding protein / Calmodulin / Cell Membrane - metabolism / Confocal microscopy / Cyclic Nucleotide-Gated Cation Channels - genetics / Cyclic Nucleotide-Gated Cation Channels - metabolism / Defects / Deoxyribonucleic acid / DNA / Fertility / fluorescence imaging cyclic nucleotide-gated channels / Gene Expression Regulation, Plant / Gene Knockout Techniques / Gene mutation / Genes / Genetic aspects / genetically modified plants / Genomes / Genomics / Germination / homozygosity / Ion channels / Kinases / Microscopy / Molecular biology / Mutation / Peptides / Physiology / Pollen / Pollen - cytology / Pollen - genetics / Pollen - metabolism / Pollen - physiology / pollen tube / Protein Transport / Proteins / Reproduction / Sperm / T-DNA / Transmission efficiency