Arabidopsis CNGC14 Mediates Calcium Influx Required for Tip Growth in Root Hairs

Root hairs create an enormous surface area to aid plants in nutrient and water acquisition, anchorage in the soil, and microbe interactions. Root hairs develop in two stages, including initiation and tip growth. Initiation occurs when a portion of genetically determined epidermal cells develop wall loosening and form a disc-shaped area of cell swelling. Tip growth happens after initiation when the root-hair “bud” elongates in a polarized fashion to eventually reach a tubular outgrowth with an apical dome (Grierson et al., 2014Grierson C. Nielsen E. Ketelaarc T. Schiefelbein J. Root hairs.Arabidopsis Book. 2014; 12: e0172Crossref Google Scholar). Polarized growth has been studied in several model systems, including the pollen tube and root-hair cells in plants, hyphae elongation in fungi, and axon extension in animal nerve systems. Calcium gradients and oscillations play a key role in controlling the direction and growth rate of these cell models. In all cases, however, the mechanism of calcium signaling has not been fully understood. In animal systems, some calcium channels have been identified that could mediate calcium fluxes (Akiyama and Kamiguchi, 2015Akiyama H. Kamiguchi H. Second messenger networks for accurate growth cone guidance.Dev. Neurobiol. 2015; 75: 411-422Crossref PubMed Scopus (14) Google Scholar). However, little is known about the Ca2+ transporters that mediate Ca2+ fluxes in plant cells. Recent studies have identified CNGC-type channels as essential for pollen tube growth and fertility (Frietsch et al., 2007Frietsch S. Wang Y.-F. Sladek C. Poulsen L.R. Romanowsky S.M. Schroeder J.I. Harper J.F. A cyclic nucleotide-gated channel is essential for polarized tip growth of pollen.Proc. Natl. Acad. Sci. USA. 2007; 104: 14531-14536Crossref PubMed Scopus (208) Google Scholar, Gao et al., 2016Gao Q.F. Gu L.L. Wang H.Q. Fei C.F. Fang X. Hussain J. Sun S.J. Dong J.Y. Liu H. Wang Y.F. Cyclic nucleotide-gated channel 18 is an essential Ca2+ channel in pollen tube tips for pollen tube guidance to ovules in Arabidopsis.Proc. Natl. Acad. Sci. USA. 2016; 113: 3096-3101Crossref PubMed Scopus (113) Google Scholar). Although a causal relationship between CNGCs and a calcium gradient in pollen tubes has yet to be established, these studies suggest that CNGCs may be a major group of channels involved in Ca2+ fluxes in plant cells. To identify the ion channels responsible for calcium influx at the tip of root hairs, we analyzed candidate transporter families and the expression patterns of individual genes. This approach led to the identification of Arabidopsis AT2G24610, which encodes cyclic nucleotide-gated channel 14 (CNGC14), as a highly expressed gene in roots. As a member of the CNGC family, the CNGC14 protein is predicted to contain six transmembrane domains, S1–S6, with a pore domain (P loop) between S5 and S6, and a C-terminal cyclic nucleotide-binding domain overlapping with a calmodulin-binding domain (reviewed in Ma and Berkowitz, 2011Ma W. Berkowitz G.A. Ca2+ conduction by plant cyclic nucleotide gated channels and associated signaling components in pathogen defense signal transduction cascades.New Phytol. 2011; 190: 566-572Crossref PubMed Scopus (97) Google Scholar). We examined the expression pattern of CNGC14 further by producing transgenic lines harboring CNGC14 promoter-GUS reporter. Histochemical analysis (Figure 1A) showed that CNGC14 was strongly expressed in root hairs and root meristem. Like many other CNGC channels, CNGC14 was localized in the plasma membrane as shown in the epidermal cells of Nicotiana benthamiana transiently transformed with 35S-GFP-CNGC14 fusion construct (Supplemental Figure 1). To determine AtCNGC14 function, we acquired and examined the phenotype of two different mutant lines of CNGC14. One was a T-DNA insertion line (SALK_206460), referred to as cngc14-1 here, which contained the T-DNA insertion in the seventh exon of the gene. The homozygous cngc14-1 lacked detectable CNGC14 transcript (Supplemental Figure 2), as analyzed by reverse transcription-PCR (RT-PCR). The other mutant is an RNA interference transgenic line expressing an artificial microRNA173 that targets CNGC14. This line, named cngc14-miR173, showed a drastically reduced level of CNGC14 transcripts and thus represented a severe knockdown line (Supplemental Figure 2). At the whole-plant level, both mutants exhibited normal growth and development throughout their life cycle when grown in the soil under normal greenhouse conditions. When the roots of wild-type and mutant seedlings grown on half-strength Murashige and Skoog medium (1/2 MS) in Petri dishes were examined, however, both the cngc14-1 and cngc14-miR173 lines had very short and sometimes branched root hairs without an apical dome, whereas they did not show defects at the root-hair initiation stage (Figure 1B). Interestingly, those short root hairs were observed when the mutant roots grew into the medium. If roots grew on the 1/2 MS medium surface (exposed to the air), we did not observe significant differences in root-hair length between mutants and wild-type plants. The short-root-hair phenotype of cngc14-1 could be complemented by expression of a transgene consisting of CNGC14 native promoter in front of its own coding region fused with YFP (Figure 1B). Furthermore, the YFP signals in the complementation lines were clearly observed at the plasma membrane of root hairs, especially on the apical dome (Figure 1C). When measuring root-hair growth, we found significantly shorter root hairs in cngc-14-1 and cngc14-miR173, but the plants harboring pCNGC14:YFP-CNGC14 in the cngc14-1 mutant background showed similar root-hair morphology to the wild-type (Supplemental Figure 2). Some CNGC members, including CNGC18, have been shown to conduct a Ca2+-permeable cation current when expressed in heterologous cells (Zhou et al., 2014Zhou L. Lan W. Jiang Y. Fang W. Luan S. A calcium-dependent protein kinase interacts with and activates a calcium channel to regulate pollen tube growth.Mol. Plant. 2014; 7: 369-376Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, Gao et al., 2016Gao Q.F. Gu L.L. Wang H.Q. Fei C.F. Fang X. Hussain J. Sun S.J. Dong J.Y. Liu H. Wang Y.F. Cyclic nucleotide-gated channel 18 is an essential Ca2+ channel in pollen tube tips for pollen tube guidance to ovules in Arabidopsis.Proc. Natl. Acad. Sci. USA. 2016; 113: 3096-3101Crossref PubMed Scopus (113) Google Scholar). We examined the channel activity of CNGC14 in Xenopus oocytes using the two-electrode voltage-clamp (TEVC) method. The TEVC recording showed that the oocytes injected with CNGC14 cRNA displayed large hyperpolarization-activated inward currents in the presence of 30 mM CaCl2, whereas the water-injected controls only showed negligible background current. It is well known that Xenopus laevis oocytes possess a calcium-activated chloride channel (CaCC), which is activated by elevated intercellular calcium. This CaCC activity has been used as a reporter for functional cloning of proteins that mediate calcium elevation (Hansen and Bräuner-Osborne, 2009Hansen K.B. Bräuner-Osborne H. Xenopus oocyte electrophysiology in GPCR drug discovery.Methods Mol. Biol. 2009; 552: 343-357Crossref PubMed Scopus (12) Google Scholar). We thus added a well-characterized CaCC inhibitor DIDS (300 μM) into the bath solution and found a strong reduction of the inward current (Figure 1D and 1E), indicating that the endogenous CaCC in Xenopus oocytes was activated, making up the major portion of the inward current recorded in CNGC14-expressing oocytes. The result suggests that CNGC14-mediated calcium influx triggers the intracellular calcium elevation, which in turn activates CaCC in the oocytes. To explore the ion selectivity of CNGC14, we found that Mg2+ but not K+, Na+, or Ba2+, elicited inward currents (Supplemental Figure 3). These results indicate that CNGC14 constitutes a calcium-permeable cation channel that has very different ionic selectivity from CNGCs in animal cells. This is consistent with results reported on other CNGCs in Arabidopsis (Gao et al., 2016Gao Q.F. Gu L.L. Wang H.Q. Fei C.F. Fang X. Hussain J. Sun S.J. Dong J.Y. Liu H. Wang Y.F. Cyclic nucleotide-gated channel 18 is an essential Ca2+ channel in pollen tube tips for pollen tube guidance to ovules in Arabidopsis.Proc. Natl. Acad. Sci. USA. 2016; 113: 3096-3101Crossref PubMed Scopus (113) Google Scholar). However, under the whole-cell recording mode, the CaCC current interferes with analysis of the calcium current. Further detailed analysis of calcium influx through CNGC14 must be pursued using the macropatch recording procedure or using expression systems that lack CaCCs. Many studies (using a vibrating probe or fluorescence resonance energy transfer [FRET] sensor Cameleon 3.6) showed that a polarized Ca2+ influx at the root-hair tip was essential for generating an apical Ca2+ gradient required for tip growth (Schiefelbein et al., 1992Schiefelbein J.W. Shipley A. Rowse P. Calcium influx at the tip of growing root-hair cells of Arabidopsis thaliana.Planta. 1992; 187: 455-459Crossref PubMed Scopus (129) Google Scholar, Monshausen et al., 2008Monshausen G.B. Messerli M.A. Gilroy S. Imaging of the Yellow Cameleon 3.6 indicator reveals that elevations in cytosolic Ca2+ follow oscillating increases in growth in root hairs of Arabidopsis.Plant Physiol. 2008; 147: 1690-1698Crossref PubMed Scopus (193) Google Scholar). CNGC14 is localized to the plasma membrane of root-hair tips and mediates Ca2+ influx in Xenopus oocytes. We thus hypothesized that CNGC14 may be the missing channel that transports Ca2+ into root hairs to generate the apical Ca2+ gradient. To test this hypothesis, we crossed the transgenic plants expressing the FRET probe, Yellow Cameleon 3.6 (YC3.6) (Monshausen et al., 2008Monshausen G.B. Messerli M.A. Gilroy S. Imaging of the Yellow Cameleon 3.6 indicator reveals that elevations in cytosolic Ca2+ follow oscillating increases in growth in root hairs of Arabidopsis.Plant Physiol. 2008; 147: 1690-1698Crossref PubMed Scopus (193) Google Scholar), with the cngc14-1 mutant. Compared with wild-type plants, the mutant plants showed an altered pattern of calcium elevation in the root-hair tips. The amplitude of calcium elevation in the cngc14-1 mutant was about 50% of that in the wild-type. The frequency of oscillations also differed between mutants (4–6 peaks per min) and the wild- type (2–4 peaks per min) in elongating root hairs (Figure 1F). Combining all the results shown here, we conclude that CNGC14 is a calcium-permeable cation channel essential for apex-focused Ca2+ changes and therefore regulates the polarized tip growth of root hairs. However, we cannot exclude the possibility that other channels in addition to CNGC14 also contribute to the calcium influx responsible for root-hair growth. We show here that the calcium signals in the root hairs of cngc14 mutants are abnormal but not completely abolished. This abnormality in the Ca2+ signature, to our understanding, is significant, because it is linked to the extensive impairment of root-hair elongation in the mutants. Therefore, we conclude that CNGC14-mediated calcium influx is essential for the root-hair tip growth under the described conditions. A recent study by Shih et al., 2015Shih H.W. DePew C.L. Miller N.D. Monshausen G.B. The cyclic nucleotide-gated channel CNGC14 regulates root gravitropism in Arabidopsis thaliana.Curr. Biol. 2015; 25: 3119-3125Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar showed that CNGC14 is involved in auxin-dependent gravitropism. The GUS expression pattern (Figure 1A) shows that CNGC14 is indeed highly expressed not only in root hairs but also in the root apex where auxin-regulated root growth takes place. In this regard, another study (Rigas et al., 2013Rigas S. Ditengou F.A. Ljung K. Daras G. Tietz O. Palme K. Hatzopoulos P. Root gravitropism and root hair development constitute coupled developmental responses regulated by auxin homeostasis in the Arabidopsis root apex.New Phytol. 2013; 197: 1130-1141Crossref PubMed Scopus (97) Google Scholar) suggested that root-hair growth and gravitropism are both regulated by auxin and may be coupled to each other. It is tempting to hypothesize that CNGC14 is a critical component in auxin-regulated events in the roots. Further work is needed to dissect the mechanism of CNGC14 action in both gravitropism and root-hair morphogenesis, linking calcium signaling with auxin response. This work was supported by the National Key Research and Development Program of China (2016YFD0300102-3) and National Natural Science Foundation of China grants 31270297, 31470356 (to L.-G.L.) and a grant from the National Science Foundation IOS1339239 (to S.L.).