A Root Stem Leaf Flower Silique Senescence leaf B a b c d UBC32 ACTIN C * Supplemental Figure 1. Expression Pattern and Protein Sequence of UBC32 Homologues in Yeast, Human, and Arabidopsis. (A) Expression of the UBC32 gene in different tissues of Arabidopsis plants. Total RNA was isolated from various tissues (roots, stems, leaves, flowers, siliques, and senescent leaves) of 5-week-old wild-type plants grown under long-day growth conditions. RT-PCR was performed with either UBC32-specific primers (top gel) or actin-specific primers (bottom gel). (B) UBC32 promoter-gus expression pattern in a transgenic Arabidopsis plant. (a) One-dayold germinating seedling. (b) Two-day-old germinating seedling. (c) Three-day-old seedling. (d) Inflorescence. Bar = 1 mm. (C) Alignment of yeast UBC6, Arabidopsis UBC32, UBC33, and UBC34, and human UBE2J 1 and UBE2J2. The conserved cysteine is indicated by a star and the membranespanning domains are showed in boxes. 1
A P1 1 LBb1 P2 ubc32-1 P5 P6 309(aa) B M P1+P2 LBb1+P2 P1+P2 LBb1+P2 P3+P4 LBb1+P4 P3+P4 LBb1+P4 ubc32-2 P3 LBb1 P4 ubc32-1 ubc32-2 C P5+P6 P5+P6 P5+P6 D Overexpression lines vector2.5 3.7 5.2 6.4 6.7 9.9 10.5 12.2 UBC32 UBC32 ACTIN rrna ubc32-1 ubc32-2 E G ubc32-1 % Root elongation vs unsupplemented medium ubc32-2 ovx2.5 ovx3.7 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 100mM NaCl F * Primary Root Length (mm) 4.5 4.0 3.5 3.0 2.5 2.0 150mM NaCl ubc32-1 ubc32-2 ovx2.5 ovx3.7 1.5 1.0 0.5 0.0 0 2 4 6 8 10 12 Days ovx2.5 ovx3.7 ubc32-2 ubc32-1 H I 100% 80% KCl ubc32-1 ubc32-2 ovx2.5 ovx3.7 60% 40% Yellow Green 1/2 MS 20% 0% ubc32-1 ubc32-2 ovx2.5 ovx3.7 2
Supplemental Figure 2. UBC32 Structure, T-DNA Insertion Diagnostic PCR, and Phenotypes in Standard Conditions or under NaCl and KCl Treatment of Mutant and 35S-UBC32 Plants. (A) Schematic diagram of UBC32 structure and T-DNA diagnostic PCR and RT-PCR. Closed boxes represent exons and lines between closed boxes represent introns. P1, forward primer; P2, reverse primer; and LBb1, primer specific to the T-DNA left border. P5 and P6 primers used for RT-PCR and quantitative RT-PCR analysis. aa, amino acids. (B) Diagnostic PCR of the T-DNA inserted in 2 different loci of UBC32. DNA from homozygous insertion lines of ubc32-1 and ubc32-2 was used. M, molecular mass markers. Primers used for PCR are indicated above each lane. (C) RT-PCR analysis of the UBC32 transcripts in wild-type and T-DNA insertion mutant seedlings. The primer pairs used for RT-PCR are shown in (A). ACTIN7 was used as an internal control. (D) Expression level of UBC32 in overexpression lines (top panel). 28S rrna was used as a loading control (bottom panel). (E) Root phenotype of representative seedlings grown on 1/2 MS plates for 5 d. Bar = 1 cm. Phenotype analysis of two ubc32 mutants, wild-type, and two 35S-UBC32 plants (ovx). (F) Quantitative analysis of primary root length of wild-type, two ubc32 mutants, and 35S-UBC32 (ovx) plants on 1/2 MS medium plates. Plot results are the means SD of 3 independent experiments (n 30). (G) Root elongation on NaCl treatment. Seedling were grown for 3 days on 1/2 MS medium and then transferred to medium supplemented with 100 mm or 150 mm NaCl for 8 days and after the treatment the entire primary root was measured. Results were standardized against values from plants transferred to unsupplemented medium. Error bars represent standard deviations of the means (n 30). Asterisk denotes t test significance compared with : P < 0.05. (H) Seeds germinated and grown on 1/2 MS medium for 2 days were transferred to 1/2 MS containing 125 mm KCl (KCl) plates (top panel) or 1/2 MS control plates (bottom panel), and were grown for a further 4 days. Bar = 1 cm. (I) Quantitative analysis of phenotypes under KCl treatment. One representative of three independent experiments is shown (n 30). 3
A Germination % 100 80 60 40 20 0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 ABA ( M) ubc32-1 ubc32-2 ovx2.5 ovx3.7 B ubc32 1 ubc32 2 Col WT ovx2.5 ovx3.7 ABA 1/2MS Supplemental Figure 3. ABA Response of ubc32 Mutants and Overexpression Lines. (A) Germination in response to ABA. Seeds were germinated at 22 o C under continuous light (10 mmol/m 2 sec white light), and the germination was scored at the fourth day. The curves corresponding to ovx2.5 and ovx3.7 overlap. Percentages are means (n 60 each) of three repeats SD. (B) Growth phenotype in response to ABA. 3-day-old seedlings were transferred to 3 M ABA or control plates, and grown for another 10 days. Bar =1 cm. 4
50 DARK MERGE BRIGHT FIELD NLuc + CLuc DOA10B-NLuc + CLuc NLuc + CLuc-UBC32 DOA10B-NLuc + CLuc-UBC32 0 Supplemental Figure 4. UBC32 Interacts with DOA10B. LUC image of N. benthamiana leaves coinfiltrated with A. tumefaciens containing different combination of constructs. The left diagram indicates the leaf panels that were infiltrated with A. tumefaciens containing the different combination of the indicated constructs. Red arrows in the leaf panels indicate the position of infiltration. The pseudocolor bar shows the range of luminescence intensity in the image. CLuc-UBC32, UBC32 fused with C-terminal region of luciferase; DOA10B-NLuc, DOA10B fused with N-terminal region of luciferase. Bar = 1cm. 5
A Ws WT bri1-5 bri1-5/ubc32 B Hypocotyl Length (cm) 1.0 0.8 0.6 0.4 0.2 0.0 Ws WT bri1-5 bri1-5/ubc32 C Ws WT bri1-5 bri1-5/ubc32 D Ws WT bri1-5 bri1-5/ubc32 BRI1 tubulin E BL BES1-P Ws WT bri1-5 bri1-5/ubc32 BES1 Rubisco F Ws WT bri1-5 bri1-5/wt F2 homo bri1-5/ubc32-1 F2 bri1-5/ubc32-2 F2 6
Supplemental Figure 5. UBC32 Mutation Partially Rescues the Phenotype of bri1-5 by Protein Accumulation and Restores the BR Sensitivity of bri1-5. (A) Hypocotyl length of 4-day-old dark-grown Ws WT,, bri1-5, and bri1-5/ubc32 seedlings. (B) Statistics of the hypocotyl length of the above seedlings. Each bar represents the mean SD of 3 independent experiments (n 30). Student s t test p < 0.01. Bar =1 cm. (C) Three-week-old soil-grown plants of Ws WT,, bri1-5, and bri1-5/ubc32 (top panel, bar =1 cm). Seven-week-old mature plants of, bri1-5, and bri1-5/ubc32 grown in soil (bottom panel, bar =10 cm). (D) The accumulation of bri1-5 accumulation in Ws WT,, bri1-5, and bri1-5/ubc32 double mutants. Total proteins were extracted with 2 SDS buffer and separated by 6% SDS-polyacrylamide gel electrophoresis (PAGE). BRI1 was detected by an anti-bri1 antibody. Tubulin was used as a loading control in the lower panel. (E) BR-induced changes in the phosphorylation status of BES1. Two-week-old seedlings of Ws WT, Col WT, bri1-5, and bri1-5/ubc32 double mutants were treated with 1 µm BL for 1 h in liquid 1/2 MS medium. Total proteins were extracted with 2 SDS buffer and separated by 10% SDS-PAGE. BES1 was detected by an anti-bes1 antibody. Rubisco was used as loading control. (F) Five-week-old-soil-grown Ws WT,, bri1-5, homozygous F2 progenies of bri1-5, bri1-5 ubc32-1, and bri1-5 ubc32-2. Bar =10 cm. 7
28 o C 37 o C mps2-1 mps2-1 ubc6 (v) mps2-1 ubc6 (Ubc6p) mps2-1ubc6 (UBC32) Supplemental Figure 6. UBC32 Can Not Complement the Yeast Ubc6p. The mps2-1 cells were transformed with an empty vector, and mps2-1 ubc6 cells were transformed with an empty vector (v), or construct expressing yeast Ubc6p or UBC32, respectively. Serial dilutions were spotted onto SD-Ura plates, and then plates were incubated at 28 o C or 37 o C for 3 days to record the photograph. Bar = 0.3 cm. 8
bri1-9 bri1-9/ubc32 NaCl Supplemental Figure 7. Salt Stress Response of bri1-9 and bri1-9/ubc32 Double Mutant. Growth phenotypes of, bri1-9, and bri1-9/ubc32 double mutant seedlings grown on 1/2 MS medium with 150 mm NaCl for 1 month. Bar = 0.5 cm. 9
Supplemental Table 1. Oligonucleotide Primers Used in This Study Name Sequence Comments qubc32 qact7 UBC32 (C93S) MLO (F240L) DOA10BLuc-UP DOA10BLuc-DP UBC32Luc-UP UBC32Luc-DP Forward: CGAGGGCGGGATTTATCATGGG Reverse: GTTGCCAATGCTCAGGGTGGTAG Forward: TCCATGAAACAACTTACAACTCCATCA Reverse: CATCGTACTCACTCTTTGAAATCCACA Forward: CTTTGAAACTAACACCAAGATTTCCTTGAGCATTTCAAACTACC Reverse: GGTAGTTTGAAATGCTCAAGGAAATCTTGGTGTTAGTTTCAAAG Forward: GCAAAACAGCAAGTTCGACTTGCACAAGTACATCAAGAGG Reverse: CCTCTTGATGTACTTGTGCAAGTCGAACTTGCTGTTTTGC 5 -AGGTACCATGGAGATTTCTCCGGCGG -3 5 -TTCGTCGACGTACTCGAGATCTTCAGTG -3 5 -AGGTACCAATAAGATGGCGGATGAG-3 5 -TTCGTCGACCAGAGTTCAAGACTGATC-3 Quantification mrna level of UBC32 by real-time quantitative PCR Used as control in real-time quantitative PCR To generate UBC32 (C93S) mutation To generate MLO-12 muation Amplification of DOA10B Amplification of UBC32 10