Volume 9, Issue 11, Pages 1520-1534 (November 2016) Rice Ferredoxin-Dependent Glutamate Synthase Regulates Nitrogen–Carbon Metabolomes and Is Genetically Differentiated between japonica and indica Subspecies  Xiaolu Yang, Jinqiang Nian, Qingjun Xie, Jian Feng, Fengxia Zhang, Hongwei Jing, Jian Zhang, Guojun Dong, Yan Liang, Juli Peng, Guodong Wang, Qian Qian, Jianru Zuo  Molecular Plant  Volume 9, Issue 11, Pages 1520-1534 (November 2016) DOI: 10.1016/j.molp.2016.09.004 Copyright © 2016 The Author Terms and Conditions

Figure 1 The abc1 Mutant Phenotype. (A) Thirteen-day-old seedlings of Nipponbare (NPB) and abc1-1 grown in field. Scale bar, 2 cm. (B) One-month-old seedlings of NPB, abc1-1, and abc1-2 grown in field. The abc1-2 mutant is seedling lethal and the plants eventually die within about a month after germination. Scale bar, 2 cm. (C) Four-month-old plants of NPB and abc1-1 grown in field. Scale bar, 10 cm. (D) Quantitative analysis of the plant height of NPB and abc1-1 plants (n = 20). (E) Quantitative analysis of the tiller number of NPB and abc1-1 plants (n = 20). (F) Top leaves derived from 1-month-old seedlings grown in field. Scale bar, 1 cm. (G) Flag leaves derived from 4-month-old plants grown in field. Scale bar, 1 cm. (H) Analysis of the chlorophyll contents in top leaves of NPB and abc1-1 plants (n = 3; biological replicates). (I) Analysis of the photosynthetic rate of NPB and abc1-1 plants (n = 4; biological replicates). Error bars in (D), (E), (H), and (I) indicate SD; **P < 0.01 (Student's t-test). Molecular Plant 2016 9, 1520-1534DOI: (10.1016/j.molp.2016.09.004) Copyright © 2016 The Author Terms and Conditions

Figure 2 The Yield-Related Traits of the abc1-1 Mutant. (A) Panicles of NPB and abc1-1. Scale bar, 5 cm. (B) Quantitative analysis of the panicle length of NPB and abc1-1 (n = 20). (C) Quantitative analysis of the primary branch number of NPB and abc1-1 (n = 20). (D) Quantitative analysis of the secondary branch number of NPB and abc1-1 (n = 20). (E) Quantitative analysis of the seed setting rate of NPB and abc1-1 (n = 20). No significant difference was observed. (F) Quantitative analysis of the grain number per panicle of NPB and abc1-1 (n = 20). (G) Seeds of NPB and abc1-1. Scale bar, 1 cm. (H) Quantitative analysis of the 1000-grain weight of NPB and abc1-1 (n = 3). Error bars in (B–F) and (H) indicate SD; **P < 0.01 (Student's t-test). Molecular Plant 2016 9, 1520-1534DOI: (10.1016/j.molp.2016.09.004) Copyright © 2016 The Author Terms and Conditions

Figure 3 Molecular Characterization of ABC1. (A) Fine mapping of the ABC1 locus. (B) The genome structure of the ABC1 gene and the mutated sites in the abc1 mutant alleles. The filled boxes, open boxes, and lines indicate exons, UTRs, and introns, respectively. The mutated nucleotide and amino acid residue of abc1-1 are shown below the genome structure and highlighted in red. The abc1-2 mutant allele contains a T-DNA insertion as indicated. (C) Four-month-old plants of NPB, abc1-1, and abc1-1 transgenic plants carrying an ABC1-FLAG transgene (abc1-1/ABC1-FLAG). Scale bar, 10 cm. (D) Flag leaves derived from plants shown in (C). Scale bar, 1 cm. (E) Quantitative analysis of the plant height of NPB, abc1-1, and abc1-1/ABC1-FLAG transgenic plants (n = 30). Transgenic line numbers are given below the graph. (F) Quantitative analysis of the tiller number of NPB, abc1-1, and abc1-1/ABC1-FLAG transgenic plants (n = 30). Transgenic line numbers are given below the graph. (G) Chlorophyll contents in top leaves of NPB, abc1-1, and abc1-1/ABC1-FLAG transgenic plants (n = 30). Transgenic line numbers are given below the graph. Error bars in (E–G) indicate SD; **P < 0.01 (Student's t-test). Molecular Plant 2016 9, 1520-1534DOI: (10.1016/j.molp.2016.09.004) Copyright © 2016 The Author Terms and Conditions

Figure 4 Functional Characterization of the Fd-GOGAT Activity. (A) Analysis of the expression level of ABC1 in NPB, abc1-1, Dongjin (DJ), and abc1-2 in leaves of 12-day-old seedlings by qRT–PCR. The relative expression level of ABC1 in wild-type (NPB or DJ) is set as 1.0. (B) Analysis of ABC1 protein levels in leaves of 12-day-old NPB and abc1-1 seedlings by immunoblotting. Quantitative analysis of the relative level is presented below the blot. (C) Analysis of the Fd-GOGAT activity in leaves of 12-day-old NPB and abc1-1 seedlings. (D) A simplified schematic map of the GS/GOGAT cycle. (E) Relative concentrations of glutamine (Gln), 2-oxoglutarate (2-OG), and glutamate (Glu) in leaves of 15-day-old NPB and abc1-1 seedlings. Error bars in (A–C) and (E) indicate SD; **P < 0.01 (Student's t-test). Molecular Plant 2016 9, 1520-1534DOI: (10.1016/j.molp.2016.09.004) Copyright © 2016 The Author Terms and Conditions

Figure 5 Phylogenetic Analysis of Fd-GOGAT Proteins in Various Species. (A) The maximum parsimony phylogenetic tree of ABC1/OsFd-GOGAT and ABC1-like proteins in various species. Bootstrap values were estimated (with 1000 replicates) to assess the relative support for each branch. The bootstrap values of 50% and above are indicated on the tree. UniProt IDs are shown. Identities (%) to Oryza sativa ABC1 sequence are listed. (B) The structural features of ABC1-like proteins. Boxes represent the domains of the enzyme. (C) Alignment of sequence involved in glutaminase activity center. Known elements important for enzyme activity and the mutant site in abc1-1 are shown. Molecular Plant 2016 9, 1520-1534DOI: (10.1016/j.molp.2016.09.004) Copyright © 2016 The Author Terms and Conditions

Figure 6 Metabolic Profiling of Key Carbon and Nitrogen Metabolites in Wild-Type and abc1-1 Plants. Metabolome analysis of carbon and nitrogen compounds in wild-type (NPB) and abc1-1 plants by gas chromatography–mass spectrometry. The aerial parts of NPB and abc1-1 seedlings at the given time points were collected and used for metabolic profiling of major compounds in carbon and nitrogen metabolism. The small boxes shown by different colors in the first and the second rows indicate the fold changes of each compound at the given time points compared with its level at day 7 in NPB and abc1-1, respectively. The small boxes in the third line indicate the fold changes of the abc1-1/NPB ratio at the given time points. Boxed numbers indicate the abc1-1/NPB ratios at day 15 with significant differences at P < 0.01 (Student's t-test). Data presented are mean values of three independent experiments. TCA, tricarboxylic acid cycle. Molecular Plant 2016 9, 1520-1534DOI: (10.1016/j.molp.2016.09.004) Copyright © 2016 The Author Terms and Conditions

Figure 7 Relative Concentrations of Selected Amino Acids and Organic Acids in the TCA Cycle. (A and B) Relative contents of selected amino acids (A) and organic acids in the TCA cycle (B) in wild-type (NPB) and abc1-1. Data presented are the same set presented in Figure 6 and are mean values of three independent experiments. Error bars indicate SD. Molecular Plant 2016 9, 1520-1534DOI: (10.1016/j.molp.2016.09.004) Copyright © 2016 The Author Terms and Conditions

Figure 8 Analysis of Single-Nucleotide Polymorphisms in ABC1 and the Domestication Patterns of ABC1. (A) Non-synonymous SNPs in ABC1. ABC1-ja, ABC1-in, and ABC1-w represent haplotypes of ABC1 in all japonica cultivars, the majority of the indica cultivars, and the haplotype mainly in wild rice, respectively. Variations in ABC1-in and ABC1-w are highlighted in red. (B) A proposed evolutionary route of ABC1. Molecular Plant 2016 9, 1520-1534DOI: (10.1016/j.molp.2016.09.004) Copyright © 2016 The Author Terms and Conditions