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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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PIN3 phosphorylation is required for PIN3 polarization and hypocotyl gravitropic response. (a) Hypocotyl bending kinetics of PIN3 mutant variants during gravitropic response. Curvatures were measured every 4 hours. Student’s T-test was calculated for the comparison of each line with the control (Col-0). PIN3-P2D mutant variant shows slower hypocotyl bending. (b–g) Localization of PIN3-YFP before and after 4 hours of gravistimulation (b,c) and after gravistimulation in PIN3-YFP-P1A (d), PIN3-YFP-P1D (e), PIN3-YFP-P2A (f), and PIN3-YFP-P2D (g). Yellow arrows indicate gravity vector. (h) Scheme of quantification showing measured membranes in hypocotyl. (i) Quantification of PIN3 signal distribution in hypocotyl endodermal cells. Student’s T-test was calculated for the comparison of outer membranes signal within each line. PIN3-P1D and PIN3-P2D exhibit higher signal intensity on outer-lateral membranes. (j) Quantitative evaluation of gravity-dependent PIN3 relocation in hypocotyl endodermal cells. Student’s T-test was calculated for the comparison of outer membranes signal within each line. Both PIN3-P1D and PIN3-P2D show defective gravity-induced PIN3-YFP relocation. (k) Localization of PIN3-YFP before and after 4 hours of 10 μM NAA treatment in wild type. White arrows indicate depletion of PIN3 protein from outer-lateral cell membranes. (l) Quantification of PIN3-YFP signal in endodermal cells of hypocotyl after 4 hours of 10 μM NAA treatment. Student’s T-test was calculated for the comparison of each line with the control (PIN3::PIN3-YFP). PIN3-P1D and PIN3-P2D exhibit reduced auxin-induced PIN3 inner-lateralization. Error bars represent SE, (**p < 0.01, ***p < 0.001). Yellow arrows indicate gravity vector. Bars = 10 µm.