Volume 6, Issue 6, Pages 1769-1780 (November 2013) A Novel Integrated Method for Large-Scale Detection, Identification, and Quantification of Widely Targeted Metabolites: Application in the Study of Rice Metabolomics  Wei Chen, Liang Gong, Zilong Guo, Wensheng Wang, Hongyan Zhang, Xianqing Liu, Sibin Yu, Lizhong Xiong, Jie Luo  Molecular Plant  Volume 6, Issue 6, Pages 1769-1780 (November 2013) DOI: 10.1093/mp/sst080 Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 1 The Main Procedures for Stepwise MIM–EPI (Multiple Ion Monitoring–Enhanced Product Ions)-Based MS2T Library Construction and MRM (Multiple Reaction Monitoring)-Based Widely Targeted Metabolic Profiling. Molecular Plant 2013 6, 1769-1780DOI: (10.1093/mp/sst080) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 2 Detection of Metabolites by MIM–EPI (Multiple Ion Monitoring–Enhanced Product Ions). (A) TIC (total ion chromatography) of EPI with 60 MIM transitions, the m/z range from 446.1 to 457.9. (B) XIC (extracted ion chromatogram) of m/z 449.1/449.1 constructed from the MIM data set. (C) MS/MS spectra of m/z 449.1 detected at 7.18min, and the metabolite was identified as luteolin-6-C-glucoside with authentic standard. Molecular Plant 2013 6, 1769-1780DOI: (10.1093/mp/sst080) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 3 The Mass Spectra and Structures of Some Newly Annotated Metabolites in Rice. (A) (a) C-pentosyl-apigenin O-p-coumaroylhexoside; (b) C-pentosyl-apigenin O-caffeoylhexoside; (c) C-pentosyl-apigenin O-feruloylhexoside; (d) C-pentosyl-chrysoeriol O-feruloylhexoside. (B) tricin O-rhamnosyl-O-malonylhexoside. (C) (a) tricin 4’-O-(syringyl alcohol) ether O-hexoside; (b) tricin 4’-O-(β-guaiacylglyceryl) ether O-hexoside. (D) Feruloyl putrescine. p-cou, p-coumaric acid; C-pen-Api DFI, diagnostic fragment ions of C-pentosyl-apigenin; hex, hexoside; caf, caffeic acid; fer, ferulic acid; C-pen-chr DFI, diagnostic fragment ions of C-pentosyl-chrysoeriol; tri-DFI, diagnostic fragment ions of tricin; mal, malonic acid; rha, rhamnose; syr alc, syringyl alcohol; β-glu gly, β-guaiacylglyceryl; put, putrescine. Molecular Plant 2013 6, 1769-1780DOI: (10.1093/mp/sst080) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 4 PCA (Principal Component Analysis) of Dehydration-Changed Metabolites in ZS97 and IRAT10. The level of significance was set at P < 0.01. PCA shows clearly separation of metabolite composition within different treatment and variety. C145N and C145D1 represent ZS97 under normal and mild dehydration stress conditions, respectively. C153N, C153D1, and C153D2 represent IRAT109 under normal, mild dehydration stress, and severe dehydration stress conditions, respectively. Mild stress, the leaves of plants under drought stress began to roll. Severe stress, the leaves of plants rolled completely and could not re-expand. Molecular Plant 2013 6, 1769-1780DOI: (10.1093/mp/sst080) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 5 Comparison of the Levels of Dehydration-Changed Metabolites in ZS97 and IRAT10. Significant metabolite changes (P < 0.01) observed for the dehydration-changed metabolome in ZS97 versus IRAT109 are shown in the metabolic map. Metabolites shown in black were detected, whereas those shown in gray were undetectable. The small boxes in the left top and bottom indicate metabolites whose levels changed under mild dehydration of ZS97 and IRAT109, respectively. The bottom-right small boxes indicate metabolites whose levels changed under severe dehydration of IRAT109. The boxes in red indicate increased and in green indicate decreased. The big boxes in the right-most indicate the different sensitivity to mild dehydration of ZS97 and IRAT109. Red font indicates ZS97 is more susceptible; green font indicates ZS97 is less susceptible. Mild stress, the leaves of plants under drought stress began to roll. Severe stress, the leaves of plants rolled completely and could not re-expand. The level of significance was set at P < 0.01. α-KG, α- ketoglutarate; Ala, alanine; Arg, arginine; Asn, asparagine; Asp, aspartate; CitA, citrate; FumA, fumarate; Glc, glucose; Glc6P, glucose-6-phosphate; Gln, glutamine; Glu, glutamate; Gly, glycine; Gpc, sn-Glycero-3-phosphocholine; His, histidine; Ile, isoleucine; Leu, leucine; LPCs, lysophosphatidylcholines; Lys, lysine; MalA, malate; Met, methionine; 5-MeoO-DMT, 5-Methoxy-N, N-dimethyltryptamine; Orn, ornithine; PA, pantothenic acid; PCr, phosphocreatine; PEP, phosphoenolpyruvate; Phe, phenylalanine; 3PGA, 3-phosphoglycerate; Pro, proline; Fer-Put, Feruloyl putrescine; Ser, serine; ShikA, shikimate; Suc, sucrose; SucA, succinate; Thr, threonine; Trg, Trigonelline; Tam, tryptamine; Trp, tryptophan; Tya, tyramine; Tyr, tyrosine; Val, valine; VB1, Vitamin B1; Api-C-H-C-H, apigenin-C-hexosyl-C-hexoside; Lut-O-H-C-H, luteolin-O-hexosyl-C-hexoside; Tri-O-sinpen, tricin O-sinapoylpentoside. Molecular Plant 2013 6, 1769-1780DOI: (10.1093/mp/sst080) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 6 PCA (Principal Component Analysis) Score Plot of 38 Accessions of Rice Germplasms According to 277 Metabolites Detected. The pale blue, blue, red, and pink dots represent the japonica, aromatic, aus, and indica varieties, respectively. Molecular Plant 2013 6, 1769-1780DOI: (10.1093/mp/sst080) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions