Volume 6, Issue 6, Pages 1945-1960 (November 2013) Control of Rice Embryo Development, Shoot Apical Meristem Maintenance, and Grain Yield by a Novel Cytochrome P450  Weibing Yang, Mingjun Gao, Xin Yin, Jiyun Liu, Yonghan Xu, Longjun Zeng, Qun Li, Shubiao Zhang, Junmin Wang, Xiaoming Zhang, Zuhua He  Molecular Plant  Volume 6, Issue 6, Pages 1945-1960 (November 2013) DOI: 10.1093/mp/sst107 Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 1 Characterization of ge Mutant. (A) Morphology of wild-type (WT) and ge seeds during grain filling stages. DAP, days after pollination. The gray dots indicate the interface of embryo and endosperm. Bar = 1mm. (B) Comparison of mature seed between WT (left) and ge (right). Bar = 1mm. (C–E) Statistical analysis of embryo area (C), endosperm area (D), and embryo/endosperm ratio (E). The data were presented as means ± SE (n > 90) and statistically analyzed by Student’s t-test (P < 0.01). Molecular Plant 2013 6, 1945-1960DOI: (10.1093/mp/sst107) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 2 Morphogenesis of Wild-Type and ge Embryos. (A–H) Sections of wild-type (WT) (A–D) and ge (E–H) embryos during developmental stages. (A, B, E, F): Bar = 50 μm. (C, D, G, H): Bar = 100 μm. (I, J) Morphology of WT (I) and ge (J) scutellum epithelial cells. Bar = 10 μm. (K, L) Morphology of WT (K) and ge (L) scutellum parenchyma cells. Bar = 10 μm. (M) Statistical analysis of scutellum parenchyma cell size (n > 150, P < 0.01). (N) Calculated number of scutellum cells (n = 8). (O, P) In situ hybridization analysis of rice histone H4 expression during WT (O) and ge (P) embryo development. 3, 5, and 7 DAP embryos: Bar = 50 μm. 10 DAP embryo: Bar = 100 μm. Molecular Plant 2013 6, 1945-1960DOI: (10.1093/mp/sst107) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 3 GE Is Required for Shoot Apical Meristem (SAM) Maintenance. (A–C) Nine-day-old seedlings of wild-type (WT) (A) and ge (B, C). White arrows indicate calluses formed in ge seedlings. Bar = 2mm. (D–F) Longitudinal sections of WT (D) and ge (E, F) seedlings. SAMs are indicated by black arrows. Bar = 100 μm. (G–I) OSH1 expression in WT (G) and ge (H, I) SAMs. SAM boundary is marked by black dots. (J–L) H4 expression in WT (J) and ge (K, L) SAMs. SAM boundary is marked by white dots. Molecular Plant 2013 6, 1945-1960DOI: (10.1093/mp/sst107) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 4 Map-Based Cloning of GE Gene and Subcellular Localization of GE Protein. (A) Segregation of large embryo and normal embryo seeds in F2 plants. (B) Seeds produced by different crosses between wild-type and ge. (C) Fine mapping of GE gene and characterization of the ge mutation. (D, E) Complementation analysis of ge mutant. (F–I) Co-localization of GE-GFP (F) and RFP-HDEL (G), an endoplasmic reticulum (ER) marker, in rice protoplast. Molecular Plant 2013 6, 1945-1960DOI: (10.1093/mp/sst107) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 5 Expression Pattern of GE. (A–E) In situ hybridization analysis of GE expression in rice developmental embryos. (F) GE expression pattern in the growing seeds of pGE::GUS transgenic plants, as revealed by GUS staining. (G) GE expression pattern in 10 DAP seed of pGE::Venus plant, as determined by YFP fluorescence microscopy analysis. Molecular Plant 2013 6, 1945-1960DOI: (10.1093/mp/sst107) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 6 GE Promoted Cell Proliferation and Plant Growth. (A) Confirmation of GE overexpression in transgenic plants by quantitative RT–PCR and RT–PCR. For quantitative RT–PCR, the rice Actin1 gene was used as an internal control. Error bars, SD (n = 3). For RT–PCR, rice UBI1was used as an internal control and 36 cycles were performed for GE with 26 cycles for UBI1. (B) Phenotype of 45-day-old wild-type (WT) and GE overexpression plants. Bar = 2cm. (C) Leaves of WT and GE overexpression plants. Bar = 1cm. (D) Morphology of leaf epidermis cells of WT and GE overexpression plants. Bar = 50 μm. (E, F) Statistical analysis of leaf length (E) (n = 24, P < 0.01) and epidermis cell length (F) (n > 300). (G) Quantitative RT–PCR analysis of cell cycle marker genes in leaves of WT and GE overexpression plants. The rice Actin1 gene was used as an internal control. Error bars, SD (n = 3). Molecular Plant 2013 6, 1945-1960DOI: (10.1093/mp/sst107) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 7 GE Overexpression Enhanced Rice Grain Yield. (A, B) Morphology of wild-type (WT) (A) and GE OE-12 (B) spikelet hull. Bar = 1mm. (C, D) Cross-section of WT (C) and GE OE-12 (D) hull. Bar = 1mm. (E, F) Morphology of WT (E) and GE OE-12 (F) cells. Bar = 25 μm. (G) Statistical analysis of hull cell size (n > 180, P < 0.01). (H) Calculated number of hull cells (n = 8). (I, J) GE overexpression increased transcript levels of cell cycle marker genes in young panicles (2–4cm). The expression levels were normalized to rice Actin1 gene and expressed relative to the WT. (K) Grains of WT and GE overexpression plants. Bar = 1mm. (L) 1000-grain weight of WT and GE overexpression seeds (n = 24). (M–O) Quantitative RT–PCR analysis of rice yield-related gene expression, including GS5 (M), GW8/OsSPL16 (N), and OsSPL14/IPA1/WFP (O) in WT and GE-OE panicles. The rice Actin1 gene was used as an internal control. Error bars, SD (n = 3). Molecular Plant 2013 6, 1945-1960DOI: (10.1093/mp/sst107) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 8 GE Restricted Embryo Growth. (A, B) Seeds of wild-type (WT) (A) and GE OE-9 (B) plants. Bar = 1mm. (C, D) Detection of GE transcripts in WT (C) and GE OE-9 (D) by in situ hybridization. Black arrows indicate the expression of GE in scutellar epithelium. (E, F) Sections of WT (E) and GE OE-12 (F) mature embryos. Bar = 100 μm. (G–I) Statistical analysis of embryo area (G), endosperm area (H), and embryo/endosperm ratio (I) (n > 80, P < 0.01). Molecular Plant 2013 6, 1945-1960DOI: (10.1093/mp/sst107) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 9 Analysis of CYP78A10 in Arabidopsis. (A) Phylogenetic analysis of GE and Arabidopsis CYP78A family members. Numbers in branches indicate bootstrap values (percent). (B) CYP78A10 expression in seeds of pCYP78A10::Venus transgenic plants. Bar = 100 μm. (C) Expression levels of CYP78A10 in different transgenic lines by quantitative RT–PCR analysis. The Arabidopsis Actin2 gene was used as an internal control. Error bars, SD (n = 3). (D) Seed morphology of Col-0 and CYP78A10 overexpression plants. Bar = 100 μm. (E, F) Statistical analysis of seed area (E) (n > 400, P < 001) and seed weight (F) (n = 8, P < 001). Molecular Plant 2013 6, 1945-1960DOI: (10.1093/mp/sst107) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions