Volume 8, Issue 1, Pages 111-121 (January 2015) Spatiotemporal Distribution of Phenolamides and the Genetics of Natural Variation of Hydroxycinnamoyl Spermidine in Rice  Xuekui Dong, Yanqiang Gao, Wei Chen, Wensheng Wang, Liang Gong, Xianqing Liu, Jie Luo  Molecular Plant  Volume 8, Issue 1, Pages 111-121 (January 2015) DOI: 10.1016/j.molp.2014.11.003 Copyright © 2015 The Author Terms and Conditions

Figure 1 Distribution of Phenolamides in Rice Flag Leaf, Culm, Panicle, Grain, and Root. (A) Heat map visualization of the relative differences in phenolamides in five tissues of rice. Each tissue type is visualized in a single column and each phenolamide is represented by a single row. Red indicates high abundance, whereas low relative phenolamides are green (color key scale above the heat map). (B and C) Content of eight major phenolamides in the five tissues of rice. DW, dry weight. The full names for the abbreviations of the metabolites are given in Table 1. Molecular Plant 2015 8, 111-121DOI: (10.1016/j.molp.2014.11.003) Copyright © 2015 The Author Terms and Conditions

Figure 2 Accumulation Patterns of Different Phenolamides in Seedling, Leaf, Culm, Panicle, and Root at Various Developmental Stages of Rice. (A) N-Feruloyl agmatine. (B) N-p-Coumaroyl agmatine. (C) N-Feruloyl putrescine. (D) N-p-Coumaroyl putrescine. (E) N′,N″-p-Coumaroyl feruloyl spermidine. (F) N′,N″-Diferuloyl spermidine. (G) N′,N″-Disinapoyl spermidine. (H) N′,N″,N‴-Diferuloyl sinapoyl spermidine. The full names for the abbreviations of the metabolites and tissues are given in Table 1 and Supplemental Table 4, respectively. Molecular Plant 2015 8, 111-121DOI: (10.1016/j.molp.2014.11.003) Copyright © 2015 The Author Terms and Conditions

Figure 3 Natural Variation of Phenolamides with and within Rice Subspecies. (A) A heat map of the natural variation of phenolamides in 156 rice varieties. The content value of each phenolamide was normalized to complete linkage hierarchical clustering. Each rice variety is visualized in a single column and each phenolamide is represented by a single row. Red indicates high abundance, whereas low relative flavonoids are green. (B) Box plot for the content of six phenolamides between japonica (gray) and indica (white). The horizontal line represents the mean and the vertical lines mark the range from the 5th to the 95th percentile of the total data. The full names for the abbreviations of the metabolites are given in Table 1. Molecular Plant 2015 8, 111-121DOI: (10.1016/j.molp.2014.11.003) Copyright © 2015 The Author Terms and Conditions

Figure 4 Manhattan Plot Displaying the GWAS Results of the Content of Six Phenolamides. The strength of association for phenolamides is indicated as the negative logarithm of the P value for the LMM model. All metabolite–SNP associations with P values below 8.88E−08 (horizontal dotted lines) are plotted against the genome location in intervals of 1 Mb. The full names for the abbreviations of the metabolites are given in Table 1. Molecular Plant 2015 8, 111-121DOI: (10.1016/j.molp.2014.11.003) Copyright © 2015 The Author Terms and Conditions

Figure 5 Locus Interactions among Significant Loci Controlling Phenolamides Accumulation. Interactions between two major loci controlling N-p-coumaroyl spermidine (A–C) and N′,N″-diferuloyl spermidine (F–H) accumulation. Three loci act in controlling the accumulation of N-p-coumaroyl spermidine (D) and N′,N″-diferuloyl spermidine (I). Proposed models for the interaction of the three loci controlling N-p-coumaroyl spermidine (E) and N′,N″-diferuloyl spermidine (J) accumulation. The full names for the abbreviations of the metabolites are given in Table 1. Molecular Plant 2015 8, 111-121DOI: (10.1016/j.molp.2014.11.003) Copyright © 2015 The Author Terms and Conditions

Figure 6 Analysis of Candidate Genes Underlying the Natural Variation of Spermidine Conjugates. (A) Conserved amino acid sequences of the BAHD family of acyltransferases. (B) Phylogenetic analysis of BAHD acyltransferases. The neighbor-joining tree was constructed using aligned full-length amino acid sequences. Bootstrap values from 1000 replicates are indicated at each node. (Bar, 0.2 amino acid substitutions per site). GenBank accession numbers are given: MpAAT1 (AAU14879), AMAT (AAW22989), CbBEBT (AAN09796), HMT/HLT (BAD89275), CmAAT1 (CAA94432), CHAT (AAN09797), AtSDT (At2g23510), AtSCT (At2g25150), the rice gene amino acids sequences from the Rice Genome Annotation Project (http://rice.plantbiology.msu.edu/cgi-bin/gbrowse/rice/), CRE2 (AAM64817). (C) A representation of the pairwise r2 value (a measure of linkage disequilibrium) among polymorphic sites in Os12g27220 and Os12g27254, where the darkness of the color of each box corresponds to the r2 value according to the legend. For the gene model, a filled black box represents the coding sequence. The stars represent the proposed functional sites. Molecular Plant 2015 8, 111-121DOI: (10.1016/j.molp.2014.11.003) Copyright © 2015 The Author Terms and Conditions

Figure 7 Functional Validation of Os12g27220 and Os12g27254 In Vivo. Box plot for the mRNA level of Os12g27220 (A) and Os12g27254 (D) in Os12g27220 and Os12g27254 transgenic individuals, respectively. Box plot for the content of spermidine and agmatine/putrescine conjugates in Os12g27220 (B and C) and Os12g27254 (E and F) transgenic individuals, respectively. WT indicates the transgenic background variety ZH11. The metabolic data of phenolamides are means ± standard error of the mean. The full names for the abbreviations of the metabolites are given in Table 1. Molecular Plant 2015 8, 111-121DOI: (10.1016/j.molp.2014.11.003) Copyright © 2015 The Author Terms and Conditions