A Rare Allele of GS2 Enhances Grain Size and Grain Yield in Rice Jiang Hu, Yuexing Wang, Yunxia Fang, Longjun Zeng, Jie Xu, Haiping Yu, Zhenyuan Shi, Jiangjie Pan, Dong Zhang, Shujing Kang, Li Zhu, Guojun Dong, Longbiao Guo, Dali Zeng, Guangheng Zhang, Lihong Xie, Guosheng Xiong, Jiayang Li, Qian Qian  Molecular Plant  Volume 8, Issue 10, Pages 1455-1465 (October 2015) DOI: 10.1016/j.molp.2015.07.002 Copyright © 2015 The Author Terms and Conditions

Figure 1 Phenotype and Grain Yield Analysis of ZH11 and ZH11(NIL-GS2BDL). (A) Parental grain phenotypes of BDL and ZH11. Scale bar, 6 mm. (B and C) Panicle phenotypes of ZH11 and ZH11(NIL-GS2BDL). Scale bar, 5 cm. (D) ZH11 and ZH11(NIL-GS2BDL) grains. Scale bar, 1 cm. (E and F) Panicle length and number of grains in the main panicle (n = 20). (G–I) Grain length, width, and thickness (n = 40). (J and K) The 1000-grain weight and grain yield per plant (n = 20). Data are given as means ± SD. Student’s t-test was used to generate the P values; **P < 0.01. Molecular Plant 2015 8, 1455-1465DOI: (10.1016/j.molp.2015.07.002) Copyright © 2015 The Author Terms and Conditions

Figure 2 Histological Analysis of Spikelet Hulls. (A) Young spikelet hulls of ZH11 and ZH11 (NIL-GS2BDL). The red line indicates the position of the cross-section. Scale bar, 3 mm. (B) Cross-sections of spikelet hulls. Scale bar, 500 μm. (C) Magnified view of the cross-section area boxed in (B). Scale bar, 100 μm. (D–F) Comparison analysis of the total cell length, cell number, and cell width in the outer parenchyma layer (n = 10). (G and H) Scanning electron microscope analysis of the outer and inner surfaces of glumes. Scale bars, 200 μm and 100 μm. (I–L) Comparison analysis of cell length and width in outer and inner glumes (n = 100). Data are given as means ± SD. Student’s t-test was used to generate the P values; **P < 0.01. Molecular Plant 2015 8, 1455-1465DOI: (10.1016/j.molp.2015.07.002) Copyright © 2015 The Author Terms and Conditions

Figure 3 Map-Based Cloning of GS2. (A) Primary map of GS2. The numerals indicate the number of recombinants. (B) Fine-scale map of GS2 generated from 2114 homozygous plants of BC4F2. (C) Delimitation of GS2 to a 7.4-kb region. (D) Gene structure and mutation sites of GS2. Empty boxes refer to the 5′ UTR and 3′ UTR, ATG and TAG to the start codon and stop codon, black boxes to exons, and the line between the black boxes to introns. (E) The QLQ and WRC domains predicted in GS2. (F) Schematic diagram of GS2 and miR396c. Red letters indicate the mutation site in GS2 mRNA. (G) GS2 transcript levels in different organs. YL, young leaf blade; YR, young root; YC, young culm; YP5 and YP13, 5 cm and 13 cm young panicles; P, L, and C, panicle, leaf blade, and culm at heading; E, endosperm after fertilization at 5 days. Actin was used as a control (n = 3). (H) Grains from transgenic plants. Scale bar, 1 cm. (I) Expression level of GS2 in transgenic plants (n = 3). (J) Grain length in transgenic plants. (n = 10). Data are given as means ± SD. Student’s t-test was used to generate the P values; **P < 0.01. NS indicates no significant level. Molecular Plant 2015 8, 1455-1465DOI: (10.1016/j.molp.2015.07.002) Copyright © 2015 The Author Terms and Conditions

Figure 4 GS2 Is a Nuclear-Localized Transcriptional Activator. (A) Subcellular localization of GS2. (B) The full-length, N-terminal deletion, and C-terminal deletion cDNAs of GS2 were cloned into pGBKT7 such that they were fused to the GAL4 DNA-binding domain. 1-394 (GS2BDL), the full-length cDNA of GS2 from BDL. 1-394 (GS2ZH11), the full-length cDNA of GS2 from ZH11. 1-63, deletion between amino acids 64 and 394. 1-99, deletion between amino acids 100 and 394. Numbers indicate the amino acids of the truncations or deletions of GS2. Molecular Plant 2015 8, 1455-1465DOI: (10.1016/j.molp.2015.07.002) Copyright © 2015 The Author Terms and Conditions

Figure 5 GS2 Application Enhanced Grain Size and Grain Weight in Existing High-yield Varieties. (A) 93-11, 93-11(NIL-GS2BDL), WYG7, and WYG7 (NIL-GS2BDL) grains. Scale bar, 1 cm. (B–D) Grain length, grain width and grain thickness (n = 40). (E and F) The 1000-grain weight and number of grains per panicle (n = 20). (G) Actual yield per plot (n = 3). Data are given as means ± SD. Student’s t-test was used to generate the P values; *P < 0.05 and **P < 0.01. NS indicates no significant level. Molecular Plant 2015 8, 1455-1465DOI: (10.1016/j.molp.2015.07.002) Copyright © 2015 The Author Terms and Conditions

Figure 6 The Effect of the BDL Allele of GS2 on Grain Yield and Quality. (A–C) Comparison of brown grains of parents, F1, and NILs. Scale bar, 2 cm. (D) Comparison of grain yield per plant (n = 20). (E) Percentage of chalkiness (n = 3). (F) Degree of chalkiness (n = 3). Data are given as means ± SD. Student’s t-test; letters indicate significant differences, P < 0.05. Molecular Plant 2015 8, 1455-1465DOI: (10.1016/j.molp.2015.07.002) Copyright © 2015 The Author Terms and Conditions

Figure 7 Comparison of Traits between PA64S/9311 and PA64S/93-11(NIL-GS2BDL). (A) Comparison of brown grains of PA64S, PA64S/93-11, and PA64S/93-11(NIL-GS2BDL). Scale bar, 2 cm. (B–D) Grain length, grain width, and grain thickness (n = 40). (E and F) The 1000-grain weight and grain yield per plant (n = 20). (G) Actual yield per plot (n = 3). (H–L) Percentage and degree of chalkiness, amylose content, alkali spreading value (gelatinization temperature), and gel consistency (n = 3). Data are given as means ± SD. Student’s t-test was used to generate the P values; *P < 0.05 and **P < 0.01, respectively. NS indicates no significant level. Molecular Plant 2015 8, 1455-1465DOI: (10.1016/j.molp.2015.07.002) Copyright © 2015 The Author Terms and Conditions