Induction of Somatic Embryos in Arabidopsis Requires Local YUCCA Expression Mediated by the Down-Regulation of Ethylene Biosynthesis  Bo Bai, Ying Hua Su, Jia Yuan, Xian Sheng Zhang  Molecular Plant  Volume 6, Issue 4, Pages 1247-1260 (July 2013) DOI: 10.1093/mp/sss154 Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 1 The YUC Genes Expression and Free IAA Levels during SE Initiation. (A–F) Expression of the YUC1, YUC2, YUC4, YUC6, YUC10, and YUC11 genes shown by qRT–PCR analysis. 0 h, representing embryonic calli cultured in ECIM for 14 d; 6 h, 12 h, 24 h, and 48 h, representing embryonic calli induced in SEIM for 6 h, 12 h, 24 h, and 48 h, respectively. 48 h*, representing the continued culture of embryonic calli in ECIM for a further 48 h after 14 d. (G) Increased levels of endogenous free IAA in embryonic calli during SE initiation. Auxin removal indicates embryonic calli induced in SEIM for 48 h after the removal of 2,4-D. Auxin non-removal indicates the continued culture of embryonic calli in ECIM containing 2,4-D for a further 48 h after 14 d. Molecular Plant 2013 6, 1247-1260DOI: (10.1093/mp/sss154) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 2 Expression Patterns of pYUC4::GUS and pYUC1::GUS, and Effects of Exogenous ACC Treatment on pYUC4::GUS and pYUC1::GUS Expression during SE Initiation. (A) The pYUC4::GUS signals shown in the embryonic callus induced in SEIM for 24 h (81.4%, n = 102). (B) The pYUC4::GUS signals shown in the embryonic callus induced in SEIM for 2 d (88.5%, n = 87). (C) The pYUC4::GUS signals in the section of embryonic callus induced in SEIM for 24 h (90.5%, n = 63). (D) The pYUC4::GUS signals in the embryonic callus treated with 200 μM exogenous ACC in SEIM for 24 h (77.8%, n = 99). (E) The pYUC4::GUS signals in the embryonic callus treated with 200 μM exogenous ACC in SEIM for 2 d (75.6%, n = 90). (F) The pYUC4::GUS signals in the section of embryonic callus treated with 200 μM exogenous ACC in SEIM for 24 h (80.7%, n = 57). (G) The pYUC1::GUS signals in the embryonic callus following culture in SEIM for 24 h (83.5%, n = 97). (H) The pYUC1::GUS signals in the embryonic callus following culture in SEIM for 2 d (78.7%, n = 89). (I) The pYUC1::GUS signals in the section of embryonic calli following culture in SEIM for 24 h (75.7%, n = 70). (J) The pYUC1::GUS signals in the embryonic callus treated with 200 μM exogenous ACC in SEIM for 24 h (77.4%, n = 93). (K) The pYUC1::GUS signals in the embryonic callus treated with 200 μM exogenous ACC in SEIM for 2 d (76.9%, n = 91). (L) The pYUC1::GUS signals in the section of embryonic callus treated with 200 μM exogenous ACC in SEIM for 24 h (79.4%, n = 63). Scale bars = 0.8 μm (A, B, D, E, G, H, J, K) and 80 m (C, F, I, L). Molecular Plant 2013 6, 1247-1260DOI: (10.1093/mp/sss154) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 3 Negative Effects of Multiple Mutations of YUCs or ACC Treatment on Somatic Embryogenesis. (A) SEs from the embryonic callus of the wild-type (Control) in SEIM for 8 d. (B) A few abnormal SEs from the embryonic callus of the yuc1 yuc4 yuc10 yuc11 quadruple mutants. (C) SEs from the embryonic callus treated with 10 μM exogenous ACC. (D–G) Lower number of SEs from embryonic calli treated with 20 μM, 50 μM, 100 μM, or 150 μM exogenous ACC. (H) No SEs were induced from the embryonic callus treated with 200 μM ACC. Scale bars = 1.2 mm. Molecular Plant 2013 6, 1247-1260DOI: (10.1093/mp/sss154) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 4 Analysis of the Expression of ACS Genes and Endogenous Ethylene Production during SE Initiation. (A–C) Expression patterns of the ACS2, ACS6, and ACS8 genes shown by qRT–PCR analysis. 0 h, representing embryonic calli cultured in ECIM for 14 d; 6 h, 12 h, 24 h, and 48 h, representing embryonic calli induced in SEIM for 6 h, 12 h, 24 h, and 48 h, respectively. 48 h*, representing the continued culture of embryonic calli in ECIM for a further 48 h after 14 d. (D) Rate of endogenous ethylene release during SE initiation. Auxin removal indicates embryonic calli induced in SEIM for 48 h after the removal of 2,4-D. Auxin non-removal indicates the continued culture of embryonic calli in ECIM containing 2,4-D for a further 48 h after 14 d. Molecular Plant 2013 6, 1247-1260DOI: (10.1093/mp/sss154) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 5 Expression of YUCs in ACC-treated and ctr1-1 Calli. Expression of the YUC1, YUC2, YUC4, YUC6, YUC10, and YUC11 genes shown by qRT–PCR analysis. Control, embryonic calli cultured in ECIM and SEIM without exogenous ACC treatment; ACC, embryonic calli cultured in ECIM and SEIM containing 200 μM exogenous ACC; ctr1-1, embryonic calli of the ctr1-1 mutant. 0 h indicates embryonic calli cultured in ECIM for 14 d; 6 h, 12 h, 24 h, and 48 h indicate embryonic calli in SEIM for 6 h, 12 h, 24 h, and 48 h, respectively. Molecular Plant 2013 6, 1247-1260DOI: (10.1093/mp/sss154) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 6 Exogenous ACC Treatment Affects the Establishment of Local Auxin Distribution. (A, B) Auxin response signals indicated by expression of the DR5rev::GFP reporter in the embryonic calli induced in SEIM for 24 h ((A) 84.5%, n = 97) or 2 d ((B) 76.6%, n = 94). (C, D) DR5rev::GFP signals (arrowheads) in the embryonic calli cultured in SEIM containing 200 μM exogenous ACC for 24 h ((C) 82.7%, n = 110) or 2 d ((D) 76.7%, n = 86). Scale bars = 80 μm. Molecular Plant 2013 6, 1247-1260DOI: (10.1093/mp/sss154) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 7 Effects of the Excessive Ethylene Production in the eto1-1 Mutant on SE Initiation. (A) SEs induced from the embryonic callus of the wild-type (WT) in SEIM for 8 d. (B) Lower number of abnormal SEs were induced from the embryonic callus of the eto1-1 mutant. (C) Expression levels of YUC4 in the calli of the WT and the eto1-1 mutant. (D) YUC4 transcript signals in the embryonic callus of the WT following culture in SEIM for 24 h (77.2%, n = 70). (E) The dispersed YUC4 transcript signals in the eto1-1 callus following culture in SEIM for 24 h (72.3%, n = 65). (F) Sense probe controls of the YUC4 gene in the embryonic callus of the WT following culture in SEIM for 24 h. Scale bars = 1.2 mm (A, B), 80 μm (D–F). Molecular Plant 2013 6, 1247-1260DOI: (10.1093/mp/sss154) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 8 Effects of the Constitutive Ethylene Response in the ctr1-1 Mutant during SE Initiation. (A) SEs induced from the embryonic callus of the wild-type (WT). (B) SEs was not induced from embryonic callus of the ctr1-1 mutant. (C) The YUC4 transcript signals in the embryonic callus of the WT following culture in SEIM for 24 h (76.1%, n = 67). (D) Dispersed and weak YUC4 transcript signals in the embryonic callus of the ctr1-1 mutant following culture in SEIM for 24 h (73.6%, n = 53). (E) The YUC1 transcript signals in the embryonic callus of the WT following culture in SEIM for 24 h (67.3%, n = 55). (F) Very weak YUC1 transcript signals in the embryonic callus of the ctr1-1 mutant following culture in SEIM for 24 h (69.1%, n = 68). (G) Sense probe controls of the YUC4 gene in the embryonic callus of the WT following culture in SEIM for 24 h. (H) Sense probe controls of the YUC1 gene in the embryonic callus of the WT following culture in SEIM for 24 h. (I) Signals of DR5::GUS were restricted to the edges (arrowheads) of the WT embryonic callus in SEIM for 24 h (83.7%, n = 86). (J) Dispersed and weak signals of DR5::GUS in the embryonic callus of the ctr1-1 mutant cultured in SEIM for 24 h (78.7%, n = 89). Scale bars = 1.2 mm (A, B), 80 m (C–H), and 0.8 mm (I, J). Molecular Plant 2013 6, 1247-1260DOI: (10.1093/mp/sss154) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions