A cyclic nucleotide-gated channel (CNGC16) in pollen is critical for stress tolerance in pollen reproductive development

Abstract

Cyclic nucleotide-gated channels (CNGCs) have been implicated in diverse aspects of plant growth and development, including responses to biotic and abiotic stress, as well as pollen tube growth and fertility. Here, genetic evidence identifies CNGC16 in Arabidopsis (Arabidopsis thaliana) as critical for pollen fertility under conditions of heat stress and drought. Two independent transfer DNA disruptions of cngc16 resulted in a greater than 10-fold stress-dependent reduction in pollen fitness and seed set. This phenotype was fully rescued through pollen expression of a CNGC16 transgene, indicating that cngc16-1 and 16-2 were both loss-of-function null alleles. The most stress-sensitive period for cngc16 pollen was during germination and the initiation of pollen tube tip growth. Pollen viability assays indicate that mutant pollen are also hypersensitive to external calcium chloride, a phenomenon analogous to calcium chloride hypersensitivities observed in other cngc mutants. A heat stress was found to increase concentrations of 3',5'-cyclic guanyl monophosphate in both pollen and leaves, as detected using an antibody-binding assay. A quantitative PCR analysis indicates that cngc16 mutant pollen have attenuated expression of several heat-stress response genes, including two heat shock transcription factor genes, HsfA2 and HsfB1. Together, these results provide evidence for a heat stress response pathway in pollen that connects a cyclic nucleotide signal, a Ca(2+)-permeable ion channel, and a signaling network that activates a downstream transcriptional heat shock response.

Figure 1.

Knockout mutations for cngc16 and corresponding stress-dependent seed set phenotype. A, Schematic diagram of CNGC16 gene model (AT3G48010) and T-DNA insertion sites in cngc16-1 and cngc16-2. Positions are shown for T-DNA insertions (triangles), exons (rectangles), introns (lines), and primers (arrows). Black regions represent transmembranes (S1–S6) and pore (P) domains in the corresponding protein. Gray shading represents the cyclic nucleotide binding domain (CNBD). B, Schematic diagram of the hot day and cold night stress cycling from −1°C to 40°C and forcing the period of pollen tube growth and fertilization to overlap with suboptimal temperatures. C, Seed set analysis of cngc16 shows a near-sterile phenotype under the hot/cold stress conditions diagrammed in B. n, Number of siliques counted. Student’s t test was performed to detect significant differences between cngc16 mutants and the wild type (wt) under hot and cold stress. **Student’s t test significant at P < 0.01.

Figure 2.

Seed set and segregation analysis showing the rescue of the male sterile phenotype. A, Seed set analyses of cngc16 knockouts rescued with a CNGC18p(i)-GFP-CNGC16 construct [seed stock nos. 1646 and 1647 for cngc16-1(−/−) and cngc16-2(−/−) backgrounds, respectively] showing seed set levels equivalent to the wild type (wt) under a hot and cold stress regime (siliques counted = 5). B, Outcrosses with representative rescue lines seed stock numbers 1646 and 1647 [for cngc16-1(−/−) and cngc16-2(−/−) backgrounds, respectively]. The rescue construct CNGC18p(i)-GFP-CNGC16 was hemizygous in all crosses. A rescue experiment was repeated with similar results using two additional lines harboring the same GFP-CNGC16 under the control of the ACA9 pollen promoter (seed stock nos. 1648 and 1649; data not shown). For crosses done with a hot/cold stress, the female had a ms1-1 phenotype. After a manual fertilization, the plants were moved to a hot day/cold night stress chamber, with the entry time at approximately 3 pm and temperature at 10°C (see Figure 1B). Statistical significance was determined by Pearson’s χ² test.

Figure 3.

Viability staining showing cngc16 mutants are hypersensitive to stress. Viability was assayed using Alexander’s reagent. Pollen were harvested from plants growing under conditions of hot days and cold nights (see Figure 1B). Values represent means ± sd of three independent experiments (n = 50–100 pollen grains for each experiment). Student’s t test was done to compare the pollen viability of cngc16 mutants with wild-type (wt) plants grown under a hot and cold stress regime. **Student’s t test significant at P < 0.01.

Figure 4.

cngc16 pollen show poor growth and bursting when germinated in vitro. Pollen grains were harvested from plants grown under normal conditions. Germination and growth were allowed to proceed for approximately 12 h on a standard in vitro agar-based growth medium containing 1 mm CaCl₂. Values represent means ± sd of three to five independent experiments, each with approximately 200 pollen grains. wt, Wild type.

Figure 5.

Alexander viability staining demonstrates that cngc16 pollen are hypersensitive to elevated CaCl₂ concentrations. Pollen grains were harvested into a water suspension from plants grown under normal conditions. Aliquots were modified as indicated and incubated for 3 h at 20°C in parallel. Incubations were done in solutions corresponding to water only, standard liquid in vitro germination medium, pH 7.5 (GM), or Tris-MES buffer (pH 7.5). Solutions were amended as indicated with Ca²⁺ using CaCl₂. Alexander staining was done after 3 h by pelleting pollen and resuspending pellets in 1 mL of Alexander stain for 30 min. Within the relatively short post hydration time frame assayed, wild-type (wt) controls for each solution showed less than 0.5% pollen grain germination. Viability counts were done with a digital camera mounted on a Leica DM IRE2 microscope. Values represent means ± sd of three independent experiments, each with approximately 50 pollen grains. Student’s t test was done to compare the pollen viability of cngc16 mutants to wild-type plants incubated at the same condition. *Student’s t test significant at P < 0.05. **Student’s t test significant at P < 0.01.

Figure 6.

Quantitative PCR indicates cngc16 pollen have attenuated expression of key stress response genes. Four stress-related genes (Zat12 [AT5G59820], HsfB1 [At4g36990], HsfA2 [At2g26150], and Bag6 [At2g46240]) were tested for their steady-state transcript levels in pollen collected after the peak heat stress (Figure 1B) from hot- and cold-stressed Arabidopsis plants. Values represent means ± sd of three to five independent experiments. Student’s t tests were conducted to compare the relative fold change in mRNA abundance of the related gene in cngc16 plants with wild-type (wt) plants. **Student’s t test significant at P < 0.01.

Figure 7.

Heat stress increases the average cGMP levels in leaves and pollen. Tissues were harvested from plants before or after a 30-min heat stress at 42°C. cGMP concentrations were assayed by competitive binding assay using cGMP-horseradish peroxidase and cGMP-specific antibody. Stress-dependent increases were measured in eight independent experiments for leaf and four independent experiments for pollen. Wilcoxon rank test was used to evaluate significance (asterisk). Means are shown with se.

Figure 8.

The expression profiles of CNGC16 show preferential expression in pollen. Plant tissue types on the graph are as follows: 7-d-old wild-type roots, 7-d-old wild-type stem, 10-d-old wild-type rosette, 7-d-old wild-type seedling and wild-type seeds. Stages of male gametophyte development are as follows: MS, microspore; BC, bicellular; TC, tricellular; MP, mature pollen; 0.5h, pollen tube germinated in vitro for 30 min; 4h, pollen tube germinated in vitro for 4 h; and SIV, pollen tube germinated semi in vivo. The expression level for mature pollen was set to 100%. Expression data were extracted from the AtGenExpress database (http://jsp.weigelworld.org/expviz/expviz.jsp; Schmid et al., 2005) and the Pollen Transcriptome Navigator (http://pollen.umd.edu/), which uses Honys and Twell (2004) data for developmental stages of pollen expression profile and Qin et al. (2009) data for the in vitro- and semi in vivo-grown pollen tubes.