Supplemental Data. Zhang et al. (2013). Plant Cell /tpc

Transcription

1 SDLTXgal SDLTH SDLTHA ADSV4 BD BDP53 BD BDP53 BD BDP53 ADAt1g1567(aa36358) ADAt1g844(aa9354) ADAt1g844(aa51354) ADAt1g844(full) Prey ADAt1g844(aa11354) ADAt3g5994(aa48418) BD BDPAL2 BD BDPAL2 BD BDPAL2 Bait ADAt3g5994(aa32418) Supplemental Figure 1. Detected KFB prey proteins in the initial Y2H screening using Arabidopsis PAL2 as the bait. After primary screening, the positive yeast clones that grew on the four dropout synthetic media (SDLeu/Trp/His/Adenine) were further liquidcultured and used for prey plasmid isolation. The plasmids then were sequenced and cotransformed back to AH19 harboring the bait vector pgbkt7pal2. 1 ul of selected yeast colonies at OD 6.5 were dropped on SDLTXαgal, SDLTH, SDLTHA to confirm these interactions. Yeast AH19 containing the plasmid of pgbkt53 expressing the fusion protein of human murine p53 (a.a. 7239) and the Gal4 DNAbinding domain and plasmid of pgadt7t expressing the fusion protein of SV4 large T antigen (a.a. 8778) with the Gal4 activation domain was used as the positive control). BD: pgbkt7.

2 A PAL1GFP PAL2GFP B KFB1GFP KFB2GFP KFB5GFP GFPKFB1 GFPKFB2 GFPKFB5 Supplemental Figure 2. Subcellular localization of PAL (A) and KFBGFP (B) fusion proteins transiently expressed in tobacco epidermal cells. Bar, 4µm.

3 PAL1HACFPc nyfpmyckfb5(δf) nyfpmyccpr3(δf) Input Ab:HA Ab:Myc IP(HA) Ab:HA Ab:Myc Supplemental Figure 3. Negative control for CoIP experiments of PALKFB interaction The truncated CPR3 that removes its Fbox domain served as the negative control for CoIP. Proteins from crude lysates (Input) and HA anitibody IPed samples were loaded for the immunoblot. The PAL proteins tagged with HA and CFPc were detected by the HA antibody, and the truncated Fbox proteins tagged with Myc and nyfp were detected with the Myc antibody. The immunobands for PAL proteins are shown for each blot. Although both the truncated KFB5 and CPR3 proteins presented at low or undetectable levels in the crude lysates, after coip, the former was detected by antimyc antibody but the latter was not.

4 A IP(HA) PAL2HA KD RT 42 C 6 C C Ab:HA 7 55 PAL2HA 4 B PAL2HA KD RT 42 C 6 C C IP(HA) Ab:Ub Supplemental Figure 4. Effect of the preparation conditions on the stability of immunoprecipitated PALHA protein Proteins extracted from tobacco leaves transiently expressed with PAL2HA or infiltrated with buffer (Mock) were IPed with antiha antibody beads. 8 μl 1X SDS loading buffer were mixed with the proteinbound beads. Samples were treated for 1 minutes at room temperature (RT), 42 C, 6 C, or C, respectively. Equal amount (5μl) of protein was loaded to the SDSPAGE gel. Both antiha antibody (A) and antiubiquitin antibody (B) were used to detect PAL2HA protein. The HA IgG bands were marked with red square and degradation products of PAL2HA were marked with solid arrow head.

5 A Relative PAL1 expression C Anthocyanin content mg/g (FW) % Suc 4% Suc 1% Suc 4% Suc B PAL Ctrl. 1% Suc 4% Suc Supplemental Figure 5. Effect of exogenous sucrose supply on PAL's transcription (A), total PAL protein concentration (B), and the content of accumulated anthocyanin (C) in Arabidopsis seedlings growing on medium containing 1% or 4% sucrose. Data are the means of the three replicates with standard deviations. Ponceau S staining served as the control for the amount of protein loading (Ctrl.).

6 KFB1 KFB2 KFB5 Ub Supplemental Figure 6. Characterization of the kfb TDNA insertion mutant lines. RTPCR analysis of the gene expression of KFB1, 2 and 5 in their TDNA insertion single and triple mutant lines.

7 A B Relative activity and content (%) KFB1 OE WT KFB2 OE C Relative activity and content (%) Relative activity and content (%) WT KFB5 OE WT Supplemental Figure 7. The relative PAL activity and lignin content in the cell walls of the T1 generation of the KFB1 (A), KFB2 (B) and KFB5 (C) overexpression lines of Arabidopsis. The data are the means of 2 or 3 experimental replicates with standard deviations. The open diamond represents PAL activity; the solid diamond represents lignin content. The amount in WT was set as.

8 WT/pMDC32 KFB1OE (32) KFB2OE (85) Supplemental Figure 8. Effect of KFB overexpression on anthocyanin pigmentation in T1 Transgenic Lines. Left panel: The front view of the 4weekold transgenic and controlrosettes; Right panel: The flipside view illustrating the disappearance of anthocyanin pigment from the petioles of the KFB1 (32) and KFB2 (85) overexpression lines. Bar=5 cm

9 Ab:PAL KFB1OE KFB2OE KFB5OE Ctrl Ab:ubi Supplemental Figure 9. Gel blotting analysis on the level of endogenous PAL protein in the KFB overexpression transgenic Arabidopsis. The PAL proteins from two independent T2 transgenic lines of each KFB genes were monitored using a developed anti PAL peptide antibody. The monoubiquitin immunoblot against the antiubiquitin antibody served for protein loading amount control.

10 3 K1 K2 K3 SM WT/pMDC Absorbance at 31 nm (mau) c4h pal1 2 tt KFB1OE (32) Retention time (min) Supplemental Figure 1. UVHPLC profiles of soluble phenolics in KFB1 overexpression line (#32), compared with the control (Wt/pMDC32), pal1, c4h and tt4 mutants. K1, kaempferol 3O[6''O(rhamnosyl) glucoside] 7Orhamnoside; K2, kaempferol 3 Oglucoside 7Orhamnoside; K3 kaempferol 3Orhamnoside 7Orhamnoside; SM, sinapoyl malate.