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. 2018 Jun 28;9:2523. doi: 10.1038/s41467-018-04952-9

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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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Fig. 3 — Brassinosteroids regulate mesocotyl elongation through the phosphorylation of CYC U2 by OsGSK2. a Morphology of mesocotyls in the BR-related lines on the fifth day after germination. T65 and Ni are wild-type control for d61 and transgenic lines, respectively. Arrows indicate the coleoptilar nodes. Scale bar, 0.5 cm. b Time course of mesocotyl elongation in the BR-related lines in a. The sample number is T65 (n = 40), d61 (n = 40), Ni (n = 43), OsGSK2-OX (n = 43), OsGSK2-RNAi (n = 45). c Cell number in a vertical line of intact mesocotyls in a (n = 80). d The expression pattern of CYC U2 in the mesocotyl, as indicated by GUS expression in the pCYC U2::GUS transgenic rice. Days after seed soaking are indicated. pl plantule, ra radicle, sc scutellum, col coleoptilar node, me mesocotyl, sh shoot; the arrow indicates the joint region among the plantule, radicle, and scutellum. Scale bar, 1 mm, 0.5 mm. e Mesocotyl morphology in CYC U2 transgenic lines on the fifth day after germination. Arrows indicate the coleoptilar nodes. Scale bar, 0.5 cm. f Time course of mesocotyl growth in the plants in e. The sample number is Ni (n = 43), CYC U2-OX (n = 40), and CYC U2-RNAi (n = 48). g Cell number in a vertical line of intact mesocotyls in e (n = 70). h, i Interaction between CYC U2 and OsGSK2 in BiFC assays (h) and yeast two-hybrid assays (i). Scale bar, 100 µm. j Interaction between CYC U2 and OsGSK2 in the Co-IP assays. k OsGSK2 phosphorylates CYC U2 in vitro. l The phosphorylation of the various mutant proteins of CYC U2 by OsGSK2 in vitro. k, l Upper panel shows autoradiography. Bottom panel shows Coommassie blue staining. m The mesocotyl phenotype of the indicated lines for genetic analysis between CYC U2 and OsGSK2. Scale bar, 0.5 cm. n–o Quantification of the mesocotyls length shown in m. In n, the samples number of Ni, Ni/OsGSK2-OX, OsGSK2-OX /CYC U2m, and Ni/CYC U2m are 45, 45, 50, and 41, respectively. In o, the sample number of Ni, Ni/OsGSK2-RNAi, OsGSK2-RNAi/CYC U2-RNAi, and Ni/CYC U2-RNAi are 45, 40, 45, and 40, respectively. Error bars are SE. P value is determined by Welch’s t test with Bonferroni correction