SUPPORTING INFORMATION Comparative proteomic analysis unveils critical pathways underlying the role of nitrogen fertilizer treatment in American elderberry Bo Yang1, Andrew L. Thomas2, and C. Michael Greenlief 1, † 1Department of Chemistry, University of Missouri, Columbia, Missouri 65211, USA 2Division of Plant Sciences, Southwest Research Center, University of Missouri, Mt. Vernon, Missouri 65712, USA †Corresponding author: C. Michael Greenlief Phone: 573-882-3288; Fax: 573-882-2754; E-mail: [email protected] TABLE OF CONTENTS Figure S1. Scatter graph of relative spots volumes detected on 2-DE gels Figure S2. Representative 2-DE maps of differentially expressed proteins Figure S3. PCA analysis of differentially expressed proteins Figure S4. Mercator protein functional categorization of each genotype Table S1. Statistical analysis of differentially expressed proteins Table S2. Protein report for MALDI-TOF/TOF Table S3. Protein report for LC-MS/MS Table S4. Identification of altered proteins after 56 kg N/ha treatment Table S5. Identification of altered proteins after 112 kg N/ha treatment Table S6. Identification of altered proteins after 169 kg N/ha treatment S1 Figure S1. Scatter graph based on the ratios of relative spots volumes detected in the master gel (y-axis) and the respective replicates (x-axis). (A-D): 0, 56, 112 and 169 kg N/ha treated Adams II. (E-H): 0, 56, 112 and 169 kg N/ha treated Bob Gordon. (I-L): 0, 56, 112 and 169 kg N/ha treated Wyldewood. A B C S2 D E F G S3 H I J K S4 L S5 Figure S2. Representative 2-DE maps of proteins extracted from American elderberry leaves. In IEF, a total of 600 µg of protein was loaded onto each pH 3-10 IPG strip (13 cm, linear). 13.5% SDS-PAGE gels were used for second dimension separation. Coomassie brilliant blue G-250 was used to visualize protein spots. Two-way ANOVA analysis indicated a total of 165 spots had significant intensity changes under different N fertilizer conditions (p < 0.05, more than 2-fold). 2D gels from samples (A) 0 kg N/ha treated Bob Gordon, (B) 0 kg N/ha treated Wyldewood, (C) 0 kg N/ha treated Adams II, (D) 56 kg N/ha treated Adams II, (E) 169 kg N/ha treated Wyldewood, and (F) 112 kg N/ha treated Adams II. A S6 B S7 C D S8 E F S9 Figure S3. Principal component analysis (PCA) of the 165 differential protein expression profile across three groups (Group 1: 0 (or control) vs 56 kg N/ha; Group 2: 0 vs 112 kg N/ha; Group 3: 0 vs 169 kg N/ha). PCA reveals almost indistinguishable features of N treatment of Adams II and Wyldewood, whereas a clear separation was detected between Bob Gordon and Adams II or Wyldewood. PC1 represents the variance as a result of technical and biological variations. PC2 represents the variance as a result of applied treatment. The ratios for x and y axis indicate the percentage of variance explained by PC1 and PC2. Group 1: Group 2: S10 Group 3: S11 Figure S4. Mercator protein functional categorization of up or down-regulated differentially expressed proteins across three N treatments of each genotype analyzed. S12 Table S1. Statistical Analysis of 165 differentially expressed proteins in three groups (Group 1: 0 (or control) vs 56 kg N/ha; Group 2: 0 vs 112 kg N/ha; Group 3: 0 vs 169 kg N/ha). Group 1: b One-way d Pos. Log2(Average Ratio) Two-way ANOVA p Values a ANOVA p No. W56/W0 B56/B0 A56/A0 B0/A0 W0/A0 B0/W0 W56/A56 B56/A56 B56/W56 Values c Genotype Nitrogen Interact 2 1.19 0.18 0.57 0.45 -0.66 1.12 -0.04 0.06 0.10 0.0268 0.1120 0.0065 0.2642 3 1.16 0.40 0.28 0.40 -0.95 1.35 -0.06 0.53 0.59 0.0025 0.0020 0.0046 0.3881 4 1.41 0.79 -0.18 0.10 -1.07 1.16 0.53 1.07 0.54 0.0488 0.0812 0.0401 0.1696 6 0.22 -0.82 -0.06 -0.12 -1.35 1.23 -1.08 -0.89 0.19 0.0021 0.0006 0.1616 0.0976 8 -0.26 -0.16 0.46 -1.41 0.10 -1.51 -0.63 -2.03 -1.41 0.0024 0.0004 0.6713 0.2053 9 0.08 -0.01 -0.42 -1.06 0.09 -1.15 0.58 -0.66 -1.24 0.0192 0.0031 0.5766 0.5326 10 0.02 -0.41 -0.45 0.71 -0.36 1.07 0.11 0.75 0.64 0.0335 0.0089 0.1716 0.5415 11 -0.11 -0.64 -0.47 0.93 -0.21 1.13 0.16 0.76 0.60 0.0097 0.0040 0.0506 0.3041 13 0.90 0.03 -2.40 -1.26 -2.12 0.86 1.19 1.17 -0.02 0.0145 0.1855 0.0946 0.0067 14 0.18 -0.45 -1.08 -0.30 -1.35 1.06 -0.09 0.34 0.43 0.0008 0.0028 0.0036 0.0103 15 0.24 0.31 1.81 0.31 -0.94 1.25 -2.51 -1.19 1.32 0.0141 0.0211 0.0394 0.0664 18 0.15 -0.47 -0.03 0.55 -0.86 1.41 -0.69 0.11 0.80 0.0074 0.0015 0.3119 0.2601 19 -0.32 -0.03 -1.10 -0.69 0.44 -1.13 1.23 0.38 -0.84 0.0199 0.0083 0.0745 0.3399 20 -0.53 -0.41 0.04 -1.00 -0.55 -0.44 -1.11 -1.44 -0.33 0.0001 0.0001 0.1241 0.3325 25 -0.75 0.66 -0.39 -1.46 0.53 -1.99 0.18 -0.41 -0.59 0.0016 0.0008 0.0827 0.0342 27 -0.31 -0.31 -0.41 0.91 0.14 0.76 0.25 1.00 0.76 0.0195 0.0040 0.1280 0.9253 29 -0.24 -0.68 0.25 1.72 0.76 0.96 0.27 0.79 0.52 0.0457 0.0148 0.2724 0.3254 30 0.16 0.20 -0.28 -1.40 -0.79 -0.60 -0.36 -0.92 -0.56 0.0001 0.0001 0.7310 0.1338 32 -0.23 -0.23 -0.37 -1.13 -0.43 -0.71 -0.29 -0.99 -0.70 0.0035 0.0007 0.0845 0.6526 33 -0.79 -1.02 0.31 0.20 -0.56 0.76 -1.66 -1.14 0.52 0.0010 0.0010 0.0452 0.0113 35 0.66 0.75 -0.02 0.57 -0.46 1.03 0.22 1.34 1.12 0.0001 0.0001 0.0034 0.0229 36 -0.27 1.32 0.09 0.24 2.26 -2.02 1.90 1.48 -0.43 0.0020 0.0004 0.5040 0.1333 37 -0.52 -0.45 -0.61 0.73 0.12 0.61 0.21 0.89 0.68 0.0068 0.0036 0.0105 0.9182 38 -1.50 0.01 -0.56 -0.84 -0.14 -0.70 -1.08 -0.27 0.81 0.0106 0.0630 0.0064 0.0631 39 -1.30 -1.21 -0.79 1.09 2.00 -0.91 1.49 0.67 -0.82 0.0008 0.0012 0.0018 0.0953 40 1.42 -0.18 -0.82 1.71 -0.10 1.81 2.15 2.35 0.20 0.0000 0.0001 0.2845 0.0043 41 -0.13 0.39 -0.38 0.33 1.05 -0.72 1.29 1.09 -0.20 0.0398 0.0072 0.9800 0.4962 42 -1.02 0.53 -0.44 0.80 1.20 -0.40 0.62 1.77 1.15 0.0007 0.0006 0.2882 0.0052 43 -0.68 -1.35 -0.14 1.44 -0.07 1.51 -0.61 0.23 0.84 0.0029 0.0047 0.0114 0.0419 46 -0.27 -0.74 -0.70 -0.02 -1.02 1.00 -0.60 -0.07 0.53 0.0050 0.0070 0.0048 0.2664 47 0.67 -1.56 -1.68 0.32 -0.58 0.90 1.78 0.45 -1.32 0.0338 0.5346 0.0416 0.0192 48 -0.85 0.13 -0.35 -0.38 1.22 -1.60 0.72 0.10 -0.61 0.0306 0.0136 0.1518 0.2256 49 0.71 -0.37 -1.42 0.92 -0.24 1.15 1.89 1.97 0.08 0.0003 0.0001 0.2061 0.0062 54 -0.58 -0.61 -0.10 1.07 1.45 -0.37 0.96 0.56 -0.40 0.0003 0.0001 0.0050 0.1324 55 0.10 1.11 -0.24 -1.41 0.26 -1.67 0.60 -0.06 -0.67 0.0407 0.0117 0.4112 0.2908 S13 56 -0.57 -0.46 -1.53 -1.08 -1.35 0.28 -0.39 -0.01 0.38 0.0014 0.0078 0.0018 0.0225 57 -0.31 -0.02 0.95 1.24 1.82 -0.57 0.56 0.27 -0.28 0.0148 0.0037 0.8420 0.1670 58 0.67 -0.03 0.28 -0.36 1.22 -1.58 1.61 -0.66 -2.28 0.0428 0.0095 0.3413 0.5322 59 -0.39 -1.40 -2.82 -0.86 0.74 -1.60 3.17 0.56 -2.61 0.0090 0.0034 0.0318 0.5816 60 -0.40 1.07 -0.28 -1.88 0.67 -2.55 0.56 -0.53 -1.09 0.0221 0.0043 0.6549 0.3582 61 -0.42 0.16 0.47 -0.54 0.01 -0.55 -0.89 -0.85 0.03 0.0422 0.0198 0.5437 0.1246 62 -0.56 -1.04 -0.94 0.15 -0.34 0.49 0.04 0.05 0.02 0.0148 0.4262 0.0009 0.4397 65 -0.55 -0.03 -0.25 -0.94 0.06 -1.00 -0.24 -0.72 -0.48 0.0332 0.0115 0.1194 0.4436 66 0.41 -0.16 0.23 0.74 -0.33 1.07 -0.15 0.35 0.50 0.0350 0.0071 0.5599 0.4445 67 -1.03 -0.26 -0.10 -0.36 1.40 -1.76 0.47 -0.52 -0.99 0.0070 0.0039 0.0643 0.1062 68 -0.38 -0.84 0.23 -0.03 1.43 -1.46 0.82 -1.10 -1.92 0.0133 0.0022 0.3537 0.5490 69 -0.90 -0.53 -2.03 1.44 2.00 -0.56 3.13 2.94 -0.19 0.0054 0.0021 0.0229 0.5378 70 0.01 -0.84 -0.37 1.19 0.99 0.20 1.36 0.72 -0.64 0.0060 0.0024 0.0744 0.1607 71 -0.65 -1.11 -0.10 1.51 0.79 0.72 0.24 0.50 0.26 0.0147 0.0205 0.0204 0.1385 72 -0.11 -1.17 0.18 2.44 0.44 2.00 0.15 1.09 0.94 0.0001 0.0001 0.0148 0.0052 73 -0.30 -0.73 -0.35 0.18 -0.71 0.90 -0.67 -0.20 0.46 0.0304 0.0257 0.0271 0.4173 74 -0.26 0.42 0.25 -1.12 -1.38 0.26 -1.89 -0.95 0.95 0.0018 0.0002 0.3776 0.5685 75 -0.29 -1.29 0.15 0.66 -0.54 1.20 -0.98 -0.77 0.20 0.0009 0.0044 0.0097 0.0037 76 -0.66 -1.01 -1.06 1.77 -0.02 1.79 0.37 1.81 1.44 0.0000 0.0001 0.0001 0.0070 77 -0.37 -0.13 0.27 -0.77 -0.28 -0.49 -0.92 -1.17 -0.25 0.0002 0.0001 0.8663 0.0654 78 -0.53 -0.24 -2.68 1.06 -0.12 1.18 2.03 3.50 1.47 0.0055 0.0013 0.0676 0.5779 79 1.97 -0.34 0.27 -0.15 -4.44 4.30 -2.74 -0.76 1.98 0.0000 0.0001 0.3276 0.0135 80 -0.51 -0.04 0.17 0.89 0.18 0.70 -0.49 0.68 1.17 0.0351 0.0059 0.6536 0.6166 81 0.11 -0.66 -0.41 3.95 0.59 3.36 1.11 3.70 2.60 0.0001 0.0001 0.1465 0.1474 82 1.85 -0.33 + ∞ + ∞ + ∞ 2.22 2.11 2.14 0.03 0.0003 0.0001 0.0993 0.0270 86 -0.39 -0.46 -0.59 0.49 -0.16 0.65 0.04 0.62 0.59 0.0020 0.0018 0.0023 0.6936 S14 Group 2: b d Pos.