Supplemental Figure 1 HDA18 has an HDAC domain and therefore has concentration dependent and TSA inhibited histone deacetylase activity.

Supplemental Figure 1 HDA18 has an HDAC domain and therefore has concentration dependent and TSA inhibited histone deacetylase activity. (A) Amino acid alignment of HDA5, HDA15 and HDA18. The blue line indicates histone deacetylase domain of HDA18. Yellow line indicates NES of HDA15. Red line indicates NES of HDA18. "*" indicates positions which have a single, fully conserved residue; ":" indicates that one of the 'strong' groups is fully conserved; "." indicates that one of the 'weaker' groups is fully conserved. The sequences are colored automatically for the sequence alignment display by the default background coloring option. (B-D) Histone deacetylase activity of HDA18 is concentration dependent and TSA inhibited. (B). Nuclear extracts from hela cells of different concentrations were incubated with fluorescent substrate for 20 min at room temperature, then the reaction was stopped by adding developer and processed to fluorescence assay 3 min later. AFU, arbitrary fluorescence units. (C). Recombinant MBP-HDA18 of different concentrations were incubated with 100µm substrate as described above. MBP, COLUMN BUFFER and ASSAY BUFFER were negative controls. 1

(D). Recombinant MBP-HDA18 activity was assayed with dose-dependent Trichostatin A (TSA) inhibition (0nM, 100nM, 250nM). MBP, COLUMN BUFFER and ASSAY BUFFER were all negative controls. Data shown mean ± SD with five replicates (** p < 0.01; Student s t-test). 2

Supplemental Figure 2 Construction and analysis of different HDA18 transgenic lines. (A) Scheme of the translational EGFP fused HDA18 constructs. (B) PCR verification of the transgenic plants by detecting the resistance gene phosphinothricin acetyltransferase (Bar) from the genomic DNA. Lane 3-2, 2-3, 2-5, 2-7, 2-9: PCR products 3

from genomic DNA isolated from individual transgenic plants respectively. (C) Verification of EGFP and HDA18-EGFP expression in transgenic lines. 3-2, 2-3, 2-5, 2-7, 2-9 were different transgenic lines. Col was wild-type. NTC was non template control. GAPDH was control. Primer sequences used to detect expression of EGFP and EGFP fused HDA18 were list in Supplemental Data Set 2. (D) (Upper) Schematic diagram of the HDA18 genomics, indicating T-DNA insertion sites and PCR target region Primers sequences used to detect expression of HDA18 were list in Supplemental Data Set 2. (Lower) Scheme of the HDA18 RNAi and over-expression constructs. (E) PCR verification of the T-DNA insertion mutants. + indicates homozygote, - indicates wildtype, M indicates the DL2000 marker, the primers used are shown on the top. The primers were determined using the three-pcr method recommended by SIGnAL (http://signal.salk.edu/tdnaprimers.2.html). Primer sequences were list in Supplemental Data Set 2. (F) Expression of HDA18 in the mutants and transgenic lines. HDA18 was decreased in the mutants and increased in the over-expression lines. Digitals on the left of panel indicates the DNA regions amplified by PCR which is shown in the upper schematic diagram in Supplemental Figure 2D. (G) Immunoblot detection of HDA18 in the mutants and transgenic lines. (H) Cross section of P WER :HDA18-GUS transgenic Arabidopsis root tip. Red arrows show the GUS signal in the N position. Blue arrow shows the GUS signal in the H position which is more modest than N position. 4

Supplemental Figure 3 Introducing the P GL2 :GFP transcriptional reporters into hda18-2 mutant by genetic crosses. After genetic crosses, we examined the F2 seedlings to get the homozygote. HM indicates hda18-2 homozygote, HZ indicates hda18-2 heterozygote, and M indicates the DL2000 plus marker. The primers were determined using the three-pcr method recommended by SIGnAL (http://signal.salk.edu/tdnaprimers.2.html). Primers used were list in Supplemental Data Set 2. 5

Supplemental Figure 4 The phenotype of the transgenic lines in which the hda18 mutant is rescued is similar to HDA18 OE, and the phenotype of transgenic lines containing knocked down HDA5/HDA15/HDA18 is similar to that of hda18. (A) Scheme of the construct for phenotype rescue. (B) PCR verification of the integrated P HDA18 :HDA18 in E.coli. Lane1-8: PCR products from individual clones respectively. Negative clones in lane7-8 have no PCR products. M: DL2000 plus marker. (C) PCR verification of the transgenic plants by detecting the resistance gene phosphinothricin acetyltransferase (Bar) from the genomic DNA. Lane1-18: PCR products from genomic DNA isolated from individual transgenic plants respectively. Lane WT: No PCR products from genomic DNA isolated from wild type Arabidopsis. (D) Expression of HDA18 in the transgenic lines for phenotype rescue. Due to the over-expression levels, lines 7#-10# were not analyzed further. Data show mean ± SD with 3 replicates 6

(Differs from the wild type; ** p < 0.01; Student s t-test). (E) Cross sections of the root tip. Hair cells in the epidermis were stained blue. Arrows show the hair cells in the N position. (F-H) Analysis of amihda5/hda15 transgenic plants in hda18 background. (F). PCR verification of the transgenic plants by detecting the resistance gene phosphinothricin acetyltransferase (Bar) from the genomic DNA. (G). Verification of loss-of-function by examining RNA levels in the transgenic plants. RNA expression of HDA5, HDA15 and HDA18 were significantly decreased in the transgenic plants. Data show mean ± SD with 3 replicates (Differs from the wild type; ** p < 0.01; Student s t-test). (H). Cross sections of the root tip. Hair cells in the epidermis were stained blue. Arrows show the hair cells in the N position. (I-K) Analysis of amihda5/hda18 transgenic plants in hda15 background. (I). PCR verification of the transgenic plants by detecting the resistance gene phosphinothricin acetyltransferase (Bar) from the genomic DNA. (J). Verification of loss-of-function by examining RNA levels in the transgenic plants. RNA expression of HDA5, HDA15 and HDA18 in B4, D1 and E1 lines was significantly decreased in the transgenic plants. Data show mean ± SD with 3 replicates (Differs from the wild type; ** p < 0.01; Student s t-test). (K). Cross sections of the root tip. Hair cells in the epidermis were stained blue. Arrows show the hair cells in the N position. 7

Supplemental Figure 5 ChIP-chip to search for DNA fragments bound by HDA18. (A) To prepare HDA18 antibody, we purified recombinant HDA18 (rhda18) as antigen, and determined the concentration of protein. Truncated HDA18 was about 36.7KD. Detailed information was list in Supplemental Table 6. (B) Immunoblot detection of antibody against HDA18. Lane1, truncated rhda18. Lane 2 and 3, Full-length rhda18. (C) Immunoprecipitation with antibody against HDA18 (+) or preimmunserum (-) in Arabidposis root cell lysis, then immunoblot detection with HDA18 antibody shows the antibody can recognize endogenous HDA18 sufficiently. (D) Unbiased distribution of HDA18 binding sites on the whole genome. Genome length was 8

scaled on top. Chromosomes 1 to 5 were shown from top to bottom sequentially. Vertical lines were potential HDA18 binding sites. 9

Supplemental Figure 6 Expression of kinase genes is reduced in mutants and elevated in over-expression lines. (A) Schematic diagrams of genes bound by HDA18. All data were retrieved from http://www.arabidopsis.org/. (B) Verification of loss-of-function by examining RNA levels in the mutants of selected HDA18 targeted genes. RNA levels of At1G18890, At3G27560, At4G26270, At4G31170 and At4G31230 were significantly reduced in mutants respectively. Data for other mutants were not shown. Data show mean ± SD with 3 replicates (Differs from the wild type; ** p < 0.01; Student s t-test). (C) Introducing the P GL2 :GFP transcriptional reporters into kinase mutants by genetic crosses. After genetic crosses, we examined the F2 seedlings to get the homozygote. HM indicates 10

SALK_145390, SALK_095751C, SALK_093390C, SALK_111509 homozygote, HZ indicates SALK_145390, SALK_095751C, SALK_093390C, SALK_111509 heterozygote. The primers were determined using the three-pcr method recommended by SIGnAL (http://signal.salk.edu/tdnaprimers.2.html). Primers used were list in Supplemental Date Set 2. (D-F) Constructions of over-expression transgenic plants of At3G27560, At4G26270, At4G331170, At4G31230. (D). Schematic diagram of At3G27560, At4G26270, At4G331170 and At4G31230 over-expression vectors. (E). PCR verification of the integrated P 35S :At4G26270, P 35S :At4G31170, P 35S :At4G31230, P At3G27560 :At3G27560 in E.coli. Lane1-8 & 9-10: PCR products from individual clones respectively. M: DL2000 plus marker. (F). Over-expression of At3G27560, At4G26270, At4G31170, At4G31230 in transgenic lines respectively. One representative line was chosen for further analysis in every type of transgenic plants. Data show mean ± SD with 3 replicates (Differs from the wild type; ** p < 0.01; Student s t-test). 11

Supplemental Figure 7 Kinase genes affect expression of pattern genes in Arabidopsis root epidermis. (A) to (D) RNA levels of pattern and related genes were altered in root tips of mutants and over-expression lines of selected kinase genes. Data show mean ± SD from 4-8 independent experiments, each with 3 replicates (Differs significantly from the wild type; * p < 0.05; ** p < 0.01; Student s t test). 12

Supplemental Figure 8 SCR affects expression of pattern genes but not cellular pattern. (A) RNA levels of pattern and related genes were altered in root tips of scr. Data show mean ± SD from 3 independent experiments, each with 3 replicates (Differs significantly from the wild type; * p < 0.05; ** p < 0.01; Student s t test). (B) Cross sections of the root tip. Hair cells in the epidermis were stained blue. 13

Supplemental Figure 9 Purifying the rmbp-hda18 to perform in vitro HDAC activity assays. A truncated HDA18 protein containing the HDAC domain and an N-terminal MBP tag was purified from DE3 cells. Before carrying out HDAC activity assays, purified protein was detected on SDS-PAGE gel, and concentration was determined by comparing to BSA standard. The arrow showed rmbp-hda18. 14

Supplemental Table 1 Root-hair and non-hair cell specification in the root epidermis of wild-type, hda18, RNAi, HDA18 OE and rescue lines. Root hairs(%) Root hair cells in the H position(%) Non-hair cells in the H position(%) Root hair cells in the N position(%) Non-hair cells in the in the N position(%) Col 41.1±3.1 97.7±5.0 2.3±5.0 1.6±3.5 98.4±3.5 hda18 44.5±5.2 97.7±5.8 2.3±5.8 6.3±6.1 ** 93.7±6.1 CS847004 HDA18 43.8±4.7 96.3±7.4 3.7±7.4 5.5±7.1 * 94.5±7.1 RNAi HDA18 46.6±5.7 93.9±10.6 6.1±10.6 11.2±6.9 ** 88.8±6.9 OE/35S HDA18 46.8±5.8 95.3±7.7 4.7±7.7 12.7±8.4 ** 87.3±8.4 OE/WER hda18 46.9±3.2 97.8±4.8 2.2±4.8 9.0±7.3 ** 91.0±7.3 HDA18 5# hda18 HDA18 6# 45.8±3.7 95.0±7.9 5.0±7.9 11.0±6.3 ** 89.0±6.3 Values indicate mean ± standard deviation of at least 10 roots for each lines (Differs significantly from the wild type; * p < 0.05; ** p < 0.01; Student s t test). 15

Supplemental Table 2 Mutants screening to test participation of HDA18 and its target genes in cellular patterning of Arabidopsis root epidermis Locus number Resources Homozygosis CP* Change At5G61070 SALK_006938 HM Y CS847004 HM Y At1G15080 SALK_005663C HM ND At1G15340 CS23991 HM N CS23990 HM N At1G18890 SALK_082441 HM Y SALK_105108C HM Y SALK_111711 HM Y At1G19485 SALK_091278C HM ND SALK_076362C HM ND At1G58250 SALK_045833 HM N SALK_027365 HM ND SALK_070277 HM N At1G79000 SALK_082118 HM N SALK_136314 HM ND At3G27560 SALK_145390 HM Y SALK_152093 HM ND At4G26270 SALK_095751C HM Y SALK_021129C HM ND At4G31170 SALK_093390C HM Y SALK_099514 HM ND At4G31230 SALK_111509 HM Y At4G34110 SALK_026293 HM N At5G49470 SALK_061416 HM N At5G53890 SALK_024464C HM Y At5G58520 SALK_144785C HM N CS822777 HM ND At5G62610 SALK_142716C HM Y CS840496 HM ND *CP: cellular pattern of root epidermis Y: yes; N: no; ND: notyet determined 16

Supplemental Table 3 Root-hair and non-hair cell specification in the root epidermis of wild-type, mutants and overexpression lines of some HDA18 targeted genes Root hair(%) Root hair cells in the H position(%) Non-hair cells in the H position(%) Root hair cells in the N position(%) Non-hair cells in the H position(%) Col 41.1±3.1 97.7±5.0 2.3±5.0 1.6±3.5 98.4±3.5 at1g18890(salk_082441) 44.6±4.4 98.5±6.0 1.5±6.0 10.5±9.3 ** 89.5±9.3 at1g18890(salk_105108c) 44.7±5.1 99.4±2.7 0.6±2.7 8.8±9.8 ** 91.2±9.8 at1g19485(salk_111711) 45.7±3.3 99.2±3.2 0.8±3.2 8.9±7.5 ** 91.1±7.5 at3g27560(salk_145390) 47.7±7.3 99.4±2.7 0.6±2.7 13.7±12.5 ** 86.3±12.5 at4g26270(salk_095751c) 45.1±3.4 98.4±4.2 1.6±4.2 12.1±6.1 ** 87.9±6.1 at4g31170(salk_093390c) 44.7±3.3 98.6±4.0 1.4±4.0 7.5±7.0 ** 92.5±7.0 at4g31230(salk_111509) 57.1±7.4 98.3±4.4 1.7±4.4 31.7±10.1 ** 68.3±10.1 at5g53890(salk_024464c) 45.7±5.8 99.3±3.0 0.7±3.0 11.0±10.5 ** 89.0±10.5 at5g62610(salk_142716) 43.2±5.0 93.9±8.4 6.1±8.4 10.3±6.8 ** 89.7±6.8 P At3G27560 :At3G27560 46.1±4.9 93.5±10.1 6.5±10.1 11.5±9.5 ** 88.5±9.5 P 35S :At4G26270 41# 46.3±4.2 98.1±4.6 1.9±4.6 11.4±6.0 ** 88.6±6.0 P 35S :At4G26270 45# 46.6±3.7 1±0 0±0 5.9±4.5 * 94.1±4.5 P 35S :At4G26270 47# 48.6±3.4 1±0 0±0 12.9±7.4 ** 87.1±7.4 P 35S :At4G31170 28# 45.8±4.3 95.8±7.7 4.2±7.7 10.5±5.4 ** 89.5±5.4 P 35S :At4G31170 32# 50.0±5.9 98.7±3.9 1.3±3.9 15.4±10.4 ** 84.6±10.4 P 35S :At4G31230 61# 47.2±3.8 93.8±9.1 6.3±9.1 11.0±7.4 ** 89.0±7.4 P 35S :At4G31230 67# 47.0±4.1 96.1±6.0 3.9±6.0 11.2±5.6 ** 88.8±5.6 P 35S :At4G31230 70# 48.3±4.9 94.2±8.5 5.8±8.5 7.6±6.5 ** 92.4±6.5 Values indicate mean ± standard deviation of at least 10 roots for each lines (Differs significantly from the wild type; * p < 0.05; ** p < 0.01; Student s t test). 17

Supplemental Table 4 Gene expression information about the HDA18 targeted genes retrieved from AREX LITE (http://www.arexdb.org/) Meristematic zone 1 Meristematic zone 2 Meristematic zone 3 Meristematic zone 4 Meristematic zone 5 Meristematic zone 6 At3G27560 At4G26270 At4G31170 At4G31230 SCM non non non non non Hair hair hair hair hair hair hair hair hair hair 93.92 167.34 0.16 0.51 90.84 30.68 2.09 2.29 31.85 98.43 28.06 49.99 6.5 20.22 153.49 51.84 3.49 3.82 27.9 86.22 54.17 96.51 20.57 64.02 243.46 82.23 0.69 0.76 12.45 38.49 41.36 73.69 10.81 33.65 140.81 47.56 2.79 3.05 8.02 24.8 65.81 117.26 9.57 29.8 173.03 58.44 1.39 1.52 9.35 28.9 56.23 100.18 15.24 47.42 214.02 72.28 4.19 4.58 9.54 29.5 Average 56.59 100.83 10.48 32.60 169.28 57.17 2.44 2.67 16.52 51.06 ftest 0.230560783 0.026261884 0.032531823 0.851170062 0.027079417 ttest 0.040135065 0.056443041 0.0028567 0.777366197 0.047822768 Data were retrieved from AREX LITE: The Arabidopsis Gene Expression Database ( http://www.arexdb.org/ ). Values indicate the expression level of genes. Ftest and ttest were tools of the statistics analysis to indicate the difference between hair cells and non-hair cells. 18

Supplemental Table 5 Genome positions of T-DNA insertion from transgenic plants. Transgenic Insertion position plants HDA18 At4G18770 1 st exon RNAi 08# HDA18 At4G02940 upstream 90bp RNAi 09# HDA18 At5g59780 upstream 20 bp RNAi 15# HDA18 K9E15 67789 chrom5 BAC RNAi 19# (intergenic regions between At5G45340 and At5G45350) HDA18 At1G21250 3 rd exon OE/35S 0910# HDA18 At5G25100 7 th exon OE/35S 14# HDA18 Intergenic regions between OE/35S 21# At4G26770 and At4G26760 HDA18 At4G31560 1 st intron OE/35S 23# HDA18 At2G17800 7 th exon OE/WER 02# HDA18 N/D OE/WER 05# N/D:not available Description A member of the R2R3-MYB gene family which likely encode transcription factors. Oxidoreductase, 2OG-Fe(II) oxygenase family protein; FUNCTIONS IN: oxidoreductase activity. Encodes a putative transcription factor (MYB59). Cell wall associated kinase, function as a signaling receptor of extracellular matrix component such as oligogalacturonides. Endomembrane protein 70 protein family. At4G26760: microtubule-associated protein 65-2 (MAP65-2). At4G26770: Phosphatidate cytidylyltransferase family protein; FUNCTIONS IN: phosphatidate cytidylyltransferase activity. Encodes HCF153, a 15-KDa protein involved in the biogenesis of the cytochrome b 6 f complex. A member of ROP GTPase family. Rac-like GTP-binding protein ARAC1/ATGP2. 19

Supplemental Table 6 Procedure for generation of the HDA18 antibody Step Operation 1 Immunogenicity predictions to confirm the sequence which was used an antigen 2 HDA18 CDS 1285-2007 were fused into pet28a vector. 3 His-tagged HDA18 was expressed in Escherichia coli stain BL21, induced by IPTG at 20 for overnight. 4 Recombinant proteins were purified by Qiagen NTA Agarose beads. 5 Purified protein was employed for animal immunity. 6 Immune serum was blotted to recombinant protein and endogenous plant protein using western blot. 7 For chromatin immunoprecipitation (ChIP) use, protein extraction was prepared according to ChIP procedure, and incubated with HDA18 antibody for overnight. 8 Immunoblot was carried out to detect the enrichment of endogenous HDA18. 20

Supplemental Table 7 Antibodies used in the experiments Antibodies Manufacturers Anti-Acetyl-Histone H4 Millipore Upstate cat#06-866 Anti-Acetyl-Histone H3 Millipore Upstate cat#06-599 Anti-Acetyl-Histone H2A Millipore Upstate cat#07-376 Anti-Acetyl-Histone H2B Millipore Upstate cat#07-373 Anti-Histone H3 Millipore Upstate cat#06-755 Anti-Histone H4 Millipore Upstate cat#07-108 Anti-Histone H3 (acetyl K9) Abcam ab10812 Anti-Histone H3 (acetyl K14) Abcam ab52946 Anti-Histone H3 (acetyl K18) Abcam ab1191 Anti-acetyl-Histone (Lys23) Millipore Upstate cat#07-355 His Tag Antibody ABGENT AM1010a Anti-MBP serum Biolabs E8030s Goat Anti-Rabbit IgG,HRP-conjugate Millipore Upstate cat#12-348 21