The Cyclic Nucleotide-Gated Channel CNGC14 Regulates Root Gravitropism in Arabidopsis thaliana  Han-Wei Shih, Cody L. DePew, Nathan D. Miller, Gabriele B. Monshausen  Current Biology  Volume 25, Issue 23, Pages 3119-3125 (December 2015) DOI: 10.1016/j.cub.2015.10.025 Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 1 CNGC14 Is Required for Auxin-Induced Ion Signaling in Arabidopsis Roots (A) Abolished root surface alkalinization in cngc14 mutants treated with 1 μM auxin (indole-3-acetic acid [IAA]). pH signaling competency was restored when the cngc14-1 mutant was functionally complemented with pCNGC14::CNGC14-eGFP (CNGC14/cngc14-1). Means + or − SD of n = 6 (WT; cngc14-1) and n = 5 (cngc14-2) independent experiments are shown. See also Figures S1A–S1C. (B) Subcellular localization of CNGC14-eGFP driven by the native CNGC14 promoter and expressed in cngc14-1 mutant background. Note the weak CNGC14-eGFP signal at the plasma membrane. Data are representative of n = 14 roots. Scale bar represents 20 μm. See also Figure S1D. (C) Auxin (1 μM IAA)-induced [Ca2+]cyt increase in epidermal cells of the elongation zone is abolished in cngc14 mutants. Means + or − SD of n = 5 independent experiments for each genotype are shown. (D) Auxin-induced [Ca2+]cyt increase in cells of the elongation zone of a representative WT root. Note that the [Ca2+]cyt increase is detectable first in the root epidermis and then migrates into the stele. (E) Loss of auxin-induced Ca2+ signaling in the root elongation zone of a representative cngc14-1 mutant. (F) Cytosolic Ca2+ signaling is not impaired in cngc14 exposed to 100 μM ATP. [Ca2+]cyt was monitored in lateral root cap cells adjacent to the columella. Means + or − SD of n = 4 independent experiments for each genotype are shown. Current Biology 2015 25, 3119-3125DOI: (10.1016/j.cub.2015.10.025) Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 2 Impaired Gravitropic Ion Signaling and Bending in Roots of Arabidopsis cngc14 Mutants (A) Average root surface pH values of WT and cngc14-1 roots before and after gravistimulation 400 μm from the root tip. Arrow indicates time point when surface pH values on opposite root flanks become significantly different (p < 0.05). Means + or − SD of n = 7 roots are shown. See also Figure S2A. (B) Average gravistimulation-induced [Ca2+]cyt changes in epidermal cells on the lower flank of WT and cngc14-1 roots. Means + or − SD of five roots for each genotype are shown; for each root, at least three epidermal cells in the apical elongation zone were analyzed. Arrow indicates time point when [Ca2+]cyt in WT root significantly increases relative to the resting [Ca2+]cyt during the first 30 s of the measurement (two-sided Student’s t test; p ≤ 0.05). (C) Delayed gravitropic bending in gravistimulated cngc14-1 roots. Means + or − SD of n = 5 (WT), n = 7 (cngc14-1), and n = 4 (cngc14-2) independent experiments are shown. See also Figures S2B and S2C. (D) Root-bending rates of gravistimulated WT and cngc14 roots. Rates were calculated from tip angles shown in (C). Arrows indicate time points when bending rates become significantly different from zero (p < 0.05). (E and F) Root caps of (E) WT and (F) cngc14-1 mutant Arabidopsis seedlings stained for 5 min with Lugol’s iodine solution. Note the purple staining of starch-filled amyloplasts in the columella. Data are representative of n = 4 roots. Scale bars represent 50 μm. (G and H) Arabidopsis (G) WT and (H) cngc14-1 root stained with 100 ng ml−1 propidium iodide. Data are representative of n = 3 roots. Scale bars represent 100 μm. Current Biology 2015 25, 3119-3125DOI: (10.1016/j.cub.2015.10.025) Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 3 Auxin-Induced Inhibition of Root Growth Is CNGC14 and Ca2+ Dependent (A) Auxin triggers an immediate repression of root cell expansion throughout the elongation zone in WT roots, but not in cngc14 mutants or roots pretreated with the Ca2+ channel blocker La3+. Growth was then monitored under control conditions for 15 min before the medium was exchanged for medium containing 1 μM auxin (left and middle) or medium containing 300 μM LaCl3, followed by 300 μM LaCl3 + 1 μM auxin (right). See also Figures S3A–S3D. (B) Auxin-induced root growth inhibition measured by tracking the root tip. Arrows indicate time points when growth rates post-auxin treatment become significantly different from growth prior to treatment (p < 0.05). Means + or − SD of n = 8 (WT) and n = 5 (cngc14-1) independent experiments are shown. (C and D) Root surface pH values of WT and cngc14-1 roots treated with 1 μM auxin. (C) Average surface pH measured 400 μm from the root tip is shown. Note that the pH response of cngc14 is delayed but approximately coincident with the second phase of the WT pH response. Arrows indicate time points when the root surface post-auxin treatment becomes significantly more alkaline than surface pH prior to treatment (p < 0.05). Asterisk indicates time point when the WT root surface pH increases significantly above the average surface pH 2 or 3 min post-auxin treatment (start of second phase). The initial acidification of the root surface immediately after auxin addition in both WT and cngc14 roots is an artifact of mixing the medium. (D) Heat plot of auxin-induced surface pH changes in a representative WT and cngc14 root at every position along the length of the growing root region over time [2] is shown. Means + or − SD or representative examples of n = 5 independent experiments for each genotype are shown. See also Figure S3E. (E) Auxin-induced root surface alkalinization is not impaired in the abp1-null mutant alleles, abp1-c1 and abp1-TD1. Auxin-induced root surface alkalinization is blocked by pretreatment with the Ca2+ channel blocker La3+, indicating that auxin-induced alkalinization is also Ca2+ dependent in the apb1 mutant background. Means + or − SD of n = 4 independent experiments for each genotype are shown. Current Biology 2015 25, 3119-3125DOI: (10.1016/j.cub.2015.10.025) Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 4 Model of CNGC14-Dependent Gravitropic Signaling in Roots A simplified presentation of asymmetric auxin fluxes in gravistimulated roots shows the preferential redirection of auxin fluxes to the lower flank of a root and basipetal transport along the epidermis (1). An unknown auxin receptor (2) is proposed to activate CNGC14 (3) to mediate Ca2+ influx into the cytosol. The receptor is likely localized to the cell surface as auxin-induced [Ca2+]cyt changes do not appear to require auxin uptake into the cytoplasm (Figure S3A). Elevated [Ca2+]cyt then rapidly regulates unknown membrane transport processes (4) to increase extracellular pH (5). A CNGC14-independent signaling pathway (6), activated by the same or other auxin receptor, also causes cell wall alkalinization. Whether this alkalinization process is regulated by Ca2+ remains to be determined. The response kinetics of this second phase of alkalinization are consistent with both posttranscriptional and very fast transcriptional regulation. Extracellular alkalinization and/or other Ca2+-activated processes not described here contribute to a reduction in cell wall extensibility and concomitant reduction in cell expansion (7). Current Biology 2015 25, 3119-3125DOI: (10.1016/j.cub.2015.10.025) Copyright © 2015 Elsevier Ltd Terms and Conditions