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. 2018 Jul 6;8:10279. doi: 10.1038/s41598-018-28188-1

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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PID kinase is involved in the gravitropism and gravity-induced PIN3 relocation in root and hypocotyl. (a) Root bending assay in wild-type, 35S::PID, wag1/wag2/pid and wag1/wag2 double mutants from the wag1/wag2/pid+ population after gravistimulation. (b) Hypocotyl bending assay in in wild-type, 35S::PID and wag1/wag2 backgrounds after gravistimulation. Student’s T-tests were calculated for the comparison of each time point with the control (PIN3::PIN3-YFP). In hypocotyl, 35S::PID shows less and wag1/wag2 more gravitropic bending as compared to the control, while only roots of 35S::PID exhibited slower bending after gravistimulation. (c,d) Schemes representing cellular membranes used for quantification of PIN3-YFP protein relocation in root columella cells. Signal intensity ratio before and after gravistimulation was calculated between lower outer (light colors) and upper outer (dark colors). Signal ratio for one root was calculated as an average of signal intensity ratios. (e–j) PIN3-YFP relocation in columella before (e,g,i) and after 30 minutes of gravitropic stimulation (f,h,j) in wild type, 35S::PID and wag1/wag2. (k) Quantification of gravity-mediated PIN3-YFP relocalization in root. Signal before gravistimulation was normalized to 1. Student’s T-tests were calculated for the comparison of each time point with the control (s). 35S::PID shows less and wag1/wag2 more gravity-induced PIN3 relocation. (l) Scheme representing membranes used for quantification of PIN3-YFP protein relocation in hypocotyl endodermal cells and quantification of gravity-mediated PIN3-YFP relocalization in hypocotyls. Underlined genotypes represent samples after gravistimulation. Student’s T-tests were calculated for the comparison of outer membranes signal within each line. 35S::PID shows less gravity-induced PIN3 relocation in hypocotyl. (m–r) PIN3-YFP protein localization in hypocotyl before (m,o,q) and after 4 hours of gravitropic stimulation (n,p,r) in wild type, 35S::PID and wag1/wag2. Experiments were repeated 3 times with 10–15 roots or hypocotyls per sample. Arrowheads indicate localization of the PIN3 protein. Error bars represent SE, (*p < 0.05, **p < 0.01, ***p < 0.001). Yellow arrows indicate gravity vector. Bars = 10 µm.