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

<div><p>The <em>Arabidopsis thaliana</em> genome contains 20 CNGCs, which are proposed to encode <u>c</u>yclic <u>n</u>ucleotide <u>g</u>ated, non-selective, Ca²⁺-permeable ion <u>c</u>hannels. 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 <em>T-DNA</em> insertions were identified for both genes and used to generate plant lines in which only one of the two alleles was segregating (e.g., <em>cngc7-1+/−/cngc8-2−/−</em> and <em>cngc7-3−/−/cngc8-1</em>+/−). While normal pollen transmission was observed for single gene mutations, pollen harboring mutations in both <em>cngc7</em> and <em>8</em> were found to be male sterile (transmission efficiency reduced by more than 3000-fold). Pollen grains harboring <em>T-DNA</em> disruptions of both <em>cngc7</em> and <em>8</em> displayed a high frequency of bursting when germinated <em>in vitro.</em> The male sterile defect could be rescued through pollen expression of a <em>CNGC7</em> or <em>8</em> 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.</p> </div

Diagram of the genomic structure of <i>CNGC7</i> and <i>8</i> and related <i>T-DNA</i> insertions.

<p>A) Locations of <i>T-DNA</i> insertions are shown for <i>cngc7-1</i>, <i>cngc7-3, cngc8-1</i> and <i>cngc8-2.</i> Arrows indicate the direction of the <i>T-DNA</i> left border. Coding sequences are highlighted with expanded rectangles; lines indicate introns and flanking DNA sequences. <i>CNGC7</i> and <i>CNGC8</i> each have five exons, and encode proteins with six transmembrane spanning domains (S1-6; highlighted in black); a pore between S5 and S6 (shaded), a cyclic nucleotide binding domain (CNBD; shaded), and a calmodulin (CaM) binding site (CaMBS; shaded with gray lines) (after Köhler et al., 1999 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055277#pone.0055277-Khler1" target="_blank">[55]</a> ). B) A diagram of chromosome 1 showing the arrangement of 2 different combinations of <i>cngc7</i> and <i>8</i> alleles in which one of the two alleles is segregating with either a <i>Basta^r</i> or <i>Sulf^r</i> marker.</p

Expression profiles showing preferential pollen expression for six <i>CNCG</i>s in <i>Arabidopsis thaliana</i>.

<p>A) Relative expression levels in different tissues are shown for <i>CNGC7</i>, <i>8</i>, <i>9</i>, <i>10</i>, <i>16</i>, and <i>18</i> (AT1G15990, AT1G19780, AT4G30560, AT1G01340, AT3G48010, and AT5G14870, respectively) obtained from the Arabidopsis eFP Browser (<a href="http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi" target="_blank">http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi</a>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055277#pone.0055277-Khler1" target="_blank">[55]</a> The expression of <i>CNGC7</i> in dry seed was arbitrarily set to 1, and the rest of the data normalized accordingly. B<b>)</b> Relative expression levels of pollen expressed <i>CNGC</i>s at different stages of pollen development obtained from The Pollen Transcriptome Navigator (<a href="http://pollen.umd.edu/" target="_blank">http://pollen.umd.edu/</a>), which uses data from Honys and Twell, 2004 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055277#pone.0055277-Honys1" target="_blank">[25]</a> (left half) and Qin et al., 2009 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055277#pone.0055277-Qin2" target="_blank">[26]</a> (right half). Developmental stages are denoted as MS: microspore; BC: bicellular; TC: tricellular; MP: mature pollen; 0.5 h: pollen tube germinated <i>in vitro</i> for 30 minutes. 4 h: pollen tube germinated <i>in vitro</i> for 4 hours, and SIV: pollen tubes after semi-<i>in vivo</i> growth through a stigma. For each data set, the expression of <i>CNGC7</i> in microspore and dry pollen were arbitrarily set to 1 and rest of the data normalized accordingly.</p

Confocal microscopy showing PM localization for GFP-CNGC7, and the <i>cngc7/8</i> bursting defect.

<p>Pollen were germinated <i>in vitro</i> and imaged. DIC images are shown to the left, and corresponding confocal fluorescence micrographs to the right. A) A negative control showing a wild type pollen tube without any GFP. B and C), GFP-CNGC7 in <i>cngc7-3−/−, 8-1</i>−/− showing a tip focused PM (plasma membrane) localization at the emerging tube (B) and the tip shank during tube extension (C). D) A non-rescued pollen from <i>cngc7-3−/−, 8-1−/−</i> (segregating a GFP-CNGC7) showing a typical bursting event at germination. Scale bar = 10 µm.</p

The <i>cngc7/8</i> pollen transmission defect can be rescued by <i>CNGC7</i> and <i>8</i> transgenes.

<p>Pollen transmission efficiencies for <i>cngc7/8</i> are shown, as scored by the transmission of the <i>Sulf^r</i> marker to F1 progeny. The <i>Sulf ^r</i> marker was associated with the <i>cngc8-1</i> allele that was segregating in the parental line. The <i>cngc7-3</i> allele was homozygous. Pollen outcrosses were made by manually pollinating females that were wild type or <i>cngc7-3</i>(−/−) with equivalent results. All pollen outcrosses were done using male parents that were verified by reciprocal crosses to be hemizygous for the transgene. In these pollen outcross assays, a perfect rescue would result in 33% of the progeny carrying the <i>Sulf^r</i> marker; because only 3 of the 4 meiotic products have the potential to show transmission to F1 progeny (see text). Numbers preceded by ss (seed stock) under each bar represent independent transgenic lines used for outcrossing. Homozygous double knockout seed stocks, created by selfing or out-crossing, are identified in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055277#pone.0055277.s004" target="_blank">Figure S4</a>. Lines shown displayed typical rescue efficiencies mediated by transgene constructs for <i>GFP-7 (9p-i-GFP-CNGC7</i>), <i>FLAG-7</i> or <i>8</i> (<i>18p-i-FLAG-CNGC7 or 8</i>), <i>FLAG-7</i> or <i>8 F to W</i> (<i>18p-i-FLAG-CNGC7-F589W</i> and <i>18p-i-FLAG-CNGC8-F624W</i>). Three additional homozyogus rescued lines were obtained (not shown) using a transgene construct ps1687 <i>18-i-GFP-CNGC8</i> (ss1402, ss1404, ss1405).</p