Supplemental Figures

Supplemental Figure 1. Expression of MYB96 in epidermal cells of stemsArabidopsis plants were grown in soil for 5 weeks before harvesting plant materials. Total RNAs were extracted from the inflorescence stems and stem epidermal peels as described previously (Suh et al., 2005). The Actin7 (At5g09810) gene was used as internal control. Transcript levels were examined by qRT-PCR. Biological triplicates were averaged. Bars indicate standard error of the mean.

Suh, M.C., Samuels, A.L., Jetter, R., Kunst, L., Pollard, M., Ohlrogge, J., and Beisson, F. (2005). Cuticular lipid composition, surface structure, and gene expression in Arabidopsis stem epidermis. Plant Physiol. 139: 1649-1665.

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Supplemental Figure 2. Epicuticular wax deposition in myb96-1 stemsFour-week-old plants grown in soil were used. Epicuticular wax crystals on the inflorescence stem segments from tip to 2 cm were visualized by Nile red staining as described previously (Pighin et al., 2004).

Pighin, J.A., Zheng, H., Balakshin, L.J., Goodman, I.P., Western, T.L., Jetter, R., Kunst, L., and Samuels, A.L. (2004). Plant cuticular lipid export requires an ABC transporter. Science 306: 702-704.

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Supplemental Figure 3. Cuticular wax composition in myb96-1 leavesRosette leaves of 4-week-old plants grown in soil were used for analysis of cuticular waxcomposition and loads. Biological triplicates were averaged and statistically treated using a student t-test (*P<0.01). Bars indicate standard error of the mean. The wax loads were expressed in μg/cm2 .

C28 C30 C32 C27 C29 C31 C33C26 C28 C30 C32C24 C26 C28 C30Fatty acids Aldehydes Primary alcohols Alkanes

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Supplemental Figure 4. Cuticular wax composition in myb96-1 stemsInflorescence stems of 5-week-old plants grown in soil were used for analysis of cuticular waxcomposition and loads. Biological triplicates were averaged and statistically treated using a student t-test (*P<0.01). Bars indicate standard error of the mean. Wax loads were expressed in μg/cm2.

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Supplemental Figure 6. Expression of cutin biosynthetic genes in myb96-1D and myb96-1Two-week-old whole plants grown on MS-agar plates were used for extraction of total RNAs. Transcript levels were examined by qRT-PCR. Biological triplicates were averaged. Bars indicate standard error of the mean.

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Supplemental Figure 5. Cutin biosynthesis in myb96-1D leavesRosette leaves of 4-week-old plants grown in soil were used for measurements of composition and contents of cutin polyester monomers as described previously (Li-Beisson et al., 2010). Biological triplicates were averaged and statistically treated using a student t-test (*P<0.01). Bars indicate standard error of the mean.

Li-Beisson, Y., Shorrosh, B., Beisson, F., X. Andersson, M., Arondel, V., Bates, P., Baud, S., Bird, D., DeBono, A., Durrett, T., Franke, R., Graham, I., Katayama, K., Kelly, A., Larson, T., Markham, J. Miquel, M., Molina, I., Nishida, I., Rowland, O. Samuels, L., Schmid, K., Wada, H., Welti, R., Xu, C., Zallot, R., and Ohlrogge, J. (2010). Acyl-Lipid Metabolism. In the Arabidopsis Book, Rockville, MD: American Society of Plant Biologist.

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Supplemental Figure 7. Rate of chlorophyll leaching in myb96-1D and myb96-1 leavesRosette leaves of 2-week-old plants grown on MS-agar plates were incubated on ice for 30 min and subsequently soaked in 80 % ethanol for the indicated time periods. The extracted chlorophyll contents at individual time points were expressed as percentages of extracted chlorophyll contents at 24 h after initial immersion. Three measurements were averaged at each time points. Bars indicate standard error of the mean.

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Supplemental Figure 8. MYB96 regulation of wax biosynthetic genes in response to ABATwo-week-old plants grown on MS-agar plates were transferred to MS liquid cultures supplemented with 20 μM ABA (6 h) before harvesting whole plants for extraction of total RNAs. Transcript levels were examined by qRT-PCR. Biological triplicates were averaged. Bars indicate standard error of the mean. Note that ABA regulation of wax biosynthetic genes depends on MYB96 (A). Genes encoding lipid metabolic enzymes are also induced by ABA. However, the inductive effects of ABA are largely independent of MYB96 (B).

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Supplemental Figure 9. Effects of MYB96 induction on wax biosynthetic gene expressionTwo-week-old transgenic plants expressing the pER8-MYB96 gene fusion under the control of a β-estradiol-inducible promoter were incubated in MS liquid cultures supplemented with 10 μM β-estradiol (β). Whole plants were harvested at the indicated time points (h) after β-estradiol application for total RNA extraction. Transcript levels were examined by qRT-PCR. Biological triplicates were averaged. Statistical significance of the measurements was determined using a student t-test by comparing with M6 measurements (t-test, *P<0.01). Bars indicate standard error of the mean. M, mock.

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Supplemental Figure 10. Binding of MYB96 to the consensus sequences in the promoters of wax biosynthetic genes in vitro(A) Putative MYB-binding consensus sequences in the gene promoters. Core binding sequences are marked by bold, italic characters. (B) in vitro binding of MYB96 to the consensus sequences. The (-) lanes are controls without adding a recombinant maltose-binding protein (MBP)-MYB96 protein.

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KCS1 -1031 AATAACTAGAAAAATACTCGAAAACTAACTAGA KCS2 -2091 GTCCCTAACTACTAACTATATTCKCS6 -1548 AAGATTAACTGTATTCKCR1 -1936 AGTTGCAGTTAAGATAACTGCAGATCER1 -797 ATGCTTAGTTAAATTTCER3 -986 AGTGTAACCATCGTTGCTTCAACTGTAAAWSD1 -761 TCTGGTTTGTCCTTAATTTTAAATATGTAAACCACTLTP3 -930 GGCCACAGTTAATTAAWBC11-1 -1240 AAAAATGGTTACCGAGWBC11-2 -873 TGAATTGGTTAGAAAT

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KCS1- +

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Supplemental Figure 11. in vitro binding of MBP alone to the consensus sequencesThe (-) lanes are controls without adding MBP proteins.

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Supplemental Figure 12. Direct binding of MYB96 to the promoters of wax biosynthetic genes in vivo(A) Representation of the wax biosynthetic gene promoters. Black lines indicate the sequence regions used for ChIP assays. NB, non-binding sequence; BS, binding sequence.(B) ChIP assays with an anti-MYC antibody. The 35S:96-MYC transgenic plants grown on MS-agar plates for 3 weeks were used. The ACT2 gene was used as negative control.

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qRT-PCR primers were designed using the Primer Express Software installed into the Applied Biosystems 7500 Real-Time PCR System. The sizes of PCR products ranged from 80 to 150 nucleotides in length. F, forward primer; R, reverse primer.

Primer Usage SequenceMYB96-F qRT-PCR 5’-TGCAGTCTCGGAAGAAGGTG MYB96-R qRT-PCR 5’-CATCTCGTGGCTTTGCTCATKCS1-F qRT-PCR 5’-TCTCCTGCTACAAACCGGAAKCS1-R qRT-PCR 5’-CCGTGTCATCGGTGAATGATKCS2-F qRT-PCR 5’-CAACCTCGCTTTCCAACAAAKCS2-R qRT-PCR 5’-TCCGGTTTTCTCAAGCACTGKCS6-F qRT-PCR 5’-TATTGTCGCCGTTGAGCTTCKCS6-R qRT-PCR 5’-TTCCATGAAAGTTGCGAAGGKCR1-F qRT-PCR 5’-CAAATCACAACCCACTTGGCKCR1-R qRT-PCR 5’-AGGTTTTTGGATGGTCGGAGCER1-F qRT-PCR 5’-AGGTCGACAGGGAGACCAACCER1-R qRT-PCR 5’-ATAAGCGCTGCCATCAACACCER3/WAX2-F qRT-PCR 5’-GGAAACGCAACGTTATTGGACER3/WAX2-R qRT-PCR 5’-AGCGTAACCGTAGATCGCACLTP3-F qRT-PCR 5’-TGGCTCCATGTGCAACCTATLTP3-R qRT-PCR 5’-GGACTGGATGCATCTGCAAGPLC1-F qRT-PCR 5’-GACATGTTTTCCGTTGTCGGPLC1-R qRT-PCR 5’-AAGACAAACCGCATGAGCAGWBC11-F qRT-PCR 5’-CCTGCGTTAAGGAACCCTTCWBC11-R qRT-PCR 5’-GGATCATCGCTTGCTTCAAAHSD1-F qRT-PCR 5’-GTGCTTCCTCCGGTATAGGCHSD1-R qRT-PCR 5’-GGAGACATCAGCATGAACGGWSD1-F qRT-PCR 5’-GCTTGGTGGTTGTTTGTTGGWSD1-R qRT-PCR 5’-TCGGGTTACCCATAAGAGGGATT1-F qRT-PCR 5’-AATTCAAACCGGAGAGGTGGATT1-R qRT-PCR 5’-AGATCCTAGGTCCGGCGTTALCR-F qRT-PCR 5’-TAAAGGTCCAACGTGGCAAGLCR-R qRT-PCR 5’-CTTACGCTGAAAAAGCCACGLACS2-F qRT-PCR 5’-GTTGATCCGGGACACTGTTGLACS2-R qRT-PCR 5’-ACGTAGGTGATCCCTTGGCTeIF4a-F qRT-PCR 5’-TGACCACACAGTCTCTGCAAeIF4a-R qRT-PCR 5’-ACCAGGGAGACTTGTTGGACACT2-F qRT-PCR 5’-CCATCCTCCGTCTTGACCTT ACT2-R qRT-PCR 5’-ACTTGCCCATCGGGTAATTCMYB96-F Subcloning 5’-AAAAAGCAGGCTCGATGGGAAGACCACCTTGCMYB96-R Subcloning 5’-AGAAAGCTGGGTTCTAGAACATCCCTTCTTGTCCTMYB96(pER8)-F Subcloning 5’-GGCTCGAGATGGGAAGACCACCTTGCMYB96(pER8)-R Subcloning 5’-CCTTAATTAACTAGAACATCCCTTCTTGTCCMYB96(MBP)-F Subcloning 5’-AAAAAGCAGGCTCGATGGGAAGACCACCTTGCMYB96(MBP)-R Subcloning 5’-AGAAAGCTGGGTTTCATTCCCATTGACCTTTGTTAGTG MYB96(MYC)-F Subcloning 5’-AACTCGAGATGGGAAGACCACCTTGC MYB96(MYC)-R Subcloning 5’-CCGGCGCGCCGAACATCCCTTCTTGTCC

Supplemental Table 1. Primers used in qRT-PCR and subcloning

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BS, DNA fragments containing MYB96-binding sequences; NB, non-binding DNA fragments. F, forward primer; R, reverse primer.

Primer SequenceKCS1(BS)-F 5’-ATTTTGGACACCTTGGCACAKCS1(BS)-R 5’-TGGGCCATGGTAAATTGATGKCS1(NB)-F 5’-GCCGTTGAAGAACAAAAGCAKCS1(NB)-R 5’-CCAACCCGAGTTTTGAGGATKCS2(BS)-F 5’-ATGCTCGCTTTGATTGATGCKCS2(BS)-R 5’-ACACAAATCCGAATAGCCGAKCS2(NB)-F 5’-CCACTCTCGAGCCATCAAAGKCS2(NB)-R 5’-CCAGTCTCCAGATTGCATGGKCS6(BS)-F 5’-TGATTTTCCCTCATGCTTGCKCS6(BS)-R 5’-TGGGCCCAATTGTGTCATTKCS6(NB)-F 5’-GTCGTAACAACCCGCAAAAAKCS6(NB)-R 5’-TCGAGATGTGCAAGCATCAAKCR1(BS)-F 5’-CCAAATGTGCAGGTTGCTCTKCR1(BS)-R 5’-ACCATGCAAGTGATCTCCCAKCR1(NB)-F 5’-TGGACATAACAGGCCAAGGAKCR1(NB)-R 5’-TGCCTTTGAGCTGCTTGAACCER3(BS)-F 5’-TGAAGAAGCAAGCCACCAAGCER3(BS)-R 5’-ACCCAATTGATGCAATGGACCER3(NB)-F 5’-AACCATTGTTAGATCAAATGCGCER3(NB)-R 5’-TCTCTTTCGTCACACCGTGAT

Supplemental Table 2. Primers used in ChIP assays

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