Cyclic nucleotide-gated channel 18 functions as an essential Ca²⁺ channel for pollen germination and pollen tube growth in Arabidopsis

ABSTRACT

We recently revealed that cyclic nucleotide-gated channel 18 (CNGC18) functioned as the main Ca²⁺ channel in pollen tube tips for pollen tube guidance to ovules by regulating external Ca²⁺ influx in Arabidopsis. In this study, we found that the reduction of external Ca²⁺ concentration ([Ca²⁺]_ext) from 10 mM to 5 mM, and further to 2 mM, led to the decreases of pollen germination percentages, but led to the increases of the percentages of ruptured pollen grains and tubes, and branched pollen tubes in vitro in cngc18-17 compared with wild type. The second point mutant allele cngc18-22 showed similar phenotypes, including reduced pollen germination percentages, increased percentages of ruptured pollen tubes, but did not show obvious different percentages of ruptured pollen grains and branched pollen tubes compared with wild type. These data demonstrate that CNGC18 plays essential roles in pollen germination and tube growth as a Ca²⁺ channel in Arabidopsis.

Sperm cells of flowering plant have lost their motility, and need to be transported by the tip growth of pollen tubes to ovules for double fertilization,¹ and pollen tube growth is guided by multiple attractants from female reproductive tissues.^1,2 A cytosolic Ca²⁺ gradient in pollen tube tips plays central roles in both pollen tube growth and orientation,^3–6 and plasma membrane Ca²⁺ channels in pollen tube tips have been proposed as the main regulators for the Ca²⁺ gradient and pollen tube growth by mediating and regulating external Ca²⁺ influx. The diverse attractants from female gametophytes may guide the pollen tube growth by regulating the activity of the Ca²⁺ channels, and by consequently regulating the Ca²⁺ gradient. The expression of 8 channel genes has been detected in Arabidopsis pollen grains and tubes, including 2 glutamate receptor-like (GLR) channels (1.2 and 3.7) and 6 cyclic nucleotide-gated channels (CNGCs) (7, 8, 9, 10, 16, and 18),^7–10 and all the 8 channels have been characterized as inward Ca²⁺ channels.^10–12 We recently found that CNGC18 is the main Ca²⁺ channel in pollen tube tips for pollen tube guidance to ovules, whereas the other 7 Ca²⁺ channels play no role, or only a negligible role, if any, in pollen tube guidance in Arabidopsis.¹² Considering that the forming and polar growth of normal pollen tubes are the prerequisite for pollen tube guidance, we have proposed that CNGC18 has 2 functions in pollen tube tips.¹² First, CNGC18 mediates external Ca²⁺ influx to establish and maintain the Ca²⁺ gradient in pollen tube tips as an inward Ca²⁺ channel. This function of CNGC18 could be essential for pollen germination and the elongating polar growth of pollen tubes. The pollen grains of T-DNA knockout mutant cngc18-1 cannot germinate to normal pollen tubes,⁷ probably because of the absence of this function of CNGC18.¹² Second, CNGC18 is a key player for pollen tube guidance, and this function of CNGC18 has been confirmed and reported recently.¹²

We have performed in vitro pollen germination assay using 10 mM Ca²⁺ condition,¹² because this [Ca²⁺]_ext is the most suitable for a high in vitro pollen germination percentage in Arabidopsis.¹³ The pollen germination percentages were approximate 70% for all the 2 point mutants cngc18-17 and cngc18-22, the 2 rescued lines COM1 and COM2, and Columbia wild type in the [Ca²⁺]_ext condition.¹² To test the function of CNGC18 as a Ca²⁺ channel in pollen germination and pollen tube growth, we performed in vitro pollen germination and tube culturing experiments using different external Ca²⁺ concentrations, especially low external Ca²⁺. The experimental results showed that the pollen germination percentages were significantly reduced in cngc18-17 and cngc18-22, were slightly reduced in COM1, but were not significantly altered in COM2 and wild type, upon the reduction of [Ca²⁺]_ext from 10 mM to 5 mM, and further to 2 mM (Fig. 1A),¹² demonstrating that CNGC18 plays an essential role in pollen germination as a Ca²⁺ channel. We also observed ruptured pollen grains and pollen tubes, and branched pollen tubes (Fig. 1B). We then analyzed the [Ca²⁺]_ext dependence of pollen grain and pollen tube rupturing, as well as pollen tube branching. The experimental results showed that the percentage of ruptured pollen grains was increased significantly from 18% to 37.6% for cngc18-17 upon the reduction of [Ca²⁺]_ext from 10 mM to 2 mM, whereas the percentages of the ruptured pollen grains were not altered significantly in cngc18-22, COM1, COM2 and wild type upon the similar [Ca²⁺]_ext reduction from 10 mM to 2 mM (Fig. 1C). The percentages of ruptured pollen tubes was increased from 7% to 26% for cngc18-17, from 6% to 22% for cngc18-22, from 7% to 15% for COM1, and from 5% to 13% for COM2, upon the [Ca²⁺]_ext reduction from 10 mM to 2 mM (Fig. 1D). The percentages of branched pollen tubes was increased significantly from 7% to 17% for cngc18-17, but not altered in cngc18-22, COM1, COM2 and wild type upon the similar [Ca²⁺]_ext reduction from 10 mM to 2 mM (Fig. 1E). These phenotypes of cngc18-17 and cngc18-22 could be caused by insufficient and/or abnormal external Ca²⁺ influx for pollen grains and pollen tubes. These data demonstrate that CNGC18 plays essential roles as an inward Ca²⁺ channel in both pollen germination and the elongating polar growth of pollen tubes in Arabidopsis.

Figure 1.

CNGC18 plays essential roles in pollen germination and pollen tube growth in Arabidopsis. (A) The analysis of [Ca²⁺]_ext dependence of pollen germination. (B) Pictures showing typical normal pollen tube of Col-0, a branched pollen tube, ruptured pollen grains and a ruptured pollen tube of cngc18-17. The branched pollen tube is indicated by an arrow, and the ruptured pollen grains and pollen tube are indicated by arrowheads. Scale bars equal to 10 μm. (C-E) The analysis of [Ca²⁺]_ext dependence of pollen grain rupturing (C), pollen tube rupturing (D), and pollen tube branching (E). Three replicates were performed. Error bars represent means ± SEM. **Significant differences from Col-0 (P < 0.01).

Disclosure of potential confllicts of interest

The authors declare no conflict of interest.

Funding

This work was supported by National Natural Science Foundation of China Grant 3107139 and China Postdoctoral Science Foundation Grant 2015M571608.