Volume 10, Issue 7, Pages 975-989 (July 2017) The Genome of Medicinal Plant Macleaya cordata Provides New Insights into Benzylisoquinoline Alkaloids Metabolism  Xiubin Liu, Yisong Liu, Peng Huang, Yongshuo Ma, Zhixing Qing, Qi Tang, Huifen Cao, Pi Cheng, Yajie Zheng, Zejun Yuan, Yuan Zhou, Jinfeng Liu, Zhaoshan Tang, Yixiu Zhuo, Yancong Zhang, Linlan Yu, Jialu Huang, Peng Yang, Qiong Peng, Jinbo Zhang, Wenkai Jiang, Zhonghua Zhang, Kui Lin, Dae-Kyun Ro, Xiaoya Chen, Xingyao Xiong, Yi Shang, Sanwen Huang, Jianguo Zeng  Molecular Plant  Volume 10, Issue 7, Pages 975-989 (July 2017) DOI: 10.1016/j.molp.2017.05.007 Copyright © 2017 The Author Terms and Conditions

Figure 1 The Sanguinarine and Chelerythrine Biosynthetic Pathways in M. cordata. The [ring-13C6]-labeled structure of the compound is indicated in blue, and the deduced bypass route is indicated with a dashed orange arrow. Molecular Plant 2017 10, 975-989DOI: (10.1016/j.molp.2017.05.007) Copyright © 2017 The Author Terms and Conditions

Figure 2 Correlation between the Metabolite Distribution and Expression Profile of the Candidate Enzymes. (A) Absolute quantifications of the contents of the end products (sanguinarine and chelerythrine) and two highly accumulated intermediates (protopine and allocryptopine) in five different tissues of M. cordata. Data represent means ± SD (n = 4). (B) Expression profiles of 39 candidate enzymes are illustrated using a gradient color. Gene expression values (TPM) were scaled by log2. The expression pattern was demonstrated by R package (Pheatmap). The value 0 was arbitrarily set to the least value −5. Genes with an expected expression pattern (indicated in red) were selected in the following functional characterization. Numeric expression values of predicted genes are shown in Supplemental Table 15. Molecular Plant 2017 10, 975-989DOI: (10.1016/j.molp.2017.05.007) Copyright © 2017 The Author Terms and Conditions

Figure 3 Evolution of the M. cordata Genome and Distribution of Gene Families. (A) Venn diagram showing unique and shared gene families between and among four sequenced datasets. Basal eudicots (green), M. cordata (yellow), core eudicots (purple), and monocots (pink). Two basal eudicots (Nodus nelumbinis and M. cordata), six eudicots (Arabidopsis thaliana, Populus trichocarpa, Glycine max, Vitis vinifera, Solanum lycopersicum, and Solanum tuberosum), and two monocots (Oryza sativa and Sorghum bicolor) and Amborella trichopoda as the outgroup. (B) Phylogenetic relationship of land plants, including the representatives with sequenced genomes for major lineages. Hypothesized polyploidy events in land plant evolution are highlighted on the phylogeny using stars. The purple star indicates the evolutionary timing of a gamma polyploidy event (million years). The evolutionary timing of the whole-genome duplication event in M. cordata evolution is shown by a red star. Additional polyploidy events are indicated by green stars. (C) Distribution of 4DTV among M. cordata, Vitis vinifera, and Nodous nelumbinis in intra- and inter-genomic comparisons. The lineage nucleotide rate in M. cordata is much slower than that of V. vinifera. Molecular Plant 2017 10, 975-989DOI: (10.1016/j.molp.2017.05.007) Copyright © 2017 The Author Terms and Conditions

Figure 4 Comparison of the Catalytic Ability of P6H from Different Plant Species. (A) LC/MS profile of dihydrosanguinarine produced from the yeast strain harboring Mco11229 (black), Mco11218 (purple), EcP6H (green), or control (blue). The yeast was feeding with Cp15 (10 μM). (B) Absolute quantification of the content of target chemical produced in (A). Data represent means ± SD (n = 3). (C) Immunoblotting of the protein sample prepared from yeast harboring Mco11229, Mco11218, or EcP6H. Actin was used as a loading control. Anti-MYC antibody was used to detect P6H-MYC-tagged protein. The experiments were repeated three times with similar results. Molecular Plant 2017 10, 975-989DOI: (10.1016/j.molp.2017.05.007) Copyright © 2017 The Author Terms and Conditions

Figure 5 Detailed Biosynthetic Pathways of SAN and CHE in M. cordata. New insights in sanguinarine and chelerythrine biosynthesis in M. cordata are highlighted by green, red, and purple rectangles: Cp27 and Cp30-Cp32 were detected in plant extract for the first time; orthologs of STORR/REPI that participate in the key branch pathway to generate addictive BIAs is not annotated in the genome of M. cordata; McP6H with better catalytic performance is indicated in orange and blue. Dashed arrow indicates that the corresponding enzyme in M. cordata is still missing. Molecular Plant 2017 10, 975-989DOI: (10.1016/j.molp.2017.05.007) Copyright © 2017 The Author Terms and Conditions