Pillitteri Fig S1 - Plant Cell · TMM MUTE ERL1 ERL2 EPF1 EPF2 CLE9 ARR16 JAZ10 POLAR ACT2 WT spch scrm-D mute scrm-D gDNA (-) Supplemental Data. Pillitteri et al. (2011). 10.1105/tpc.111.088583

A B

C D

Supplemental Figure 1. Images of representative 5-dag seedlings

used in this study.(A) WT; (B) spch; (C) scrm-D; (D) scrm-D mute. Images were taken under the same magnification. Scale bar, 1

mm.

.

Supplemental Data. Pillitteri et al. (2011). 10.1105/tpc.111.088583

1

CellCell wallDevelopmentDNAProteinRNA regulation of transcription

Signaling receptor kinasesSignaling

Auxin metabolismHormone metabolism

Mitochrondrial ECT/ATP synthesis

PhotosynthesisMajor CHO metabolism

TCA/org. transformation

GlycolysisLipid metabolism

TransportSecondary metabolism

StressRedox regulation

scrm

-D m

ute

scrm

-D

spch

ratio up-/down regulated0 1 0.0001

0.05

p-value

Supplemental Figure 2. Summary of overrepresented functional gene categories associated with cell-enriched genotypes. Columns represent scrm-D, spch, or scrm-D mute as specified. Gene functional categories overrepresented in each genetic background are indicated. Gene upregulation was determined through comparison with wild-type seedling expression. Overrepresentation analysis was performed using Pageman software (Usadel et al., 2006). Usadel, B., Nagel, A., Steinhauser, D., Gibon, Y., Blasing, O.E., Redestig, H., Sreenivasulu, N., Krall, L., Hannah, M.A., Poree, F., Fernie, A.R., and Stitt, M. (2006). PageMan: an interactive ontology tool to generate, display, and annotate overview graphs for profiling experiments. BMC Bioinformatics 7, 535.


2

TMM

MUTE

ERL1ERL2EPF1EPF2CLE9

ARR16

JAZ10

POLARACT2

WT spch

scrm

-D m

ute

scrm

-DgD

NA(-)


3

POLARBASL

EPF2

TMMERECTA

ERL1ERL2

0.0 180.0

Absolute

Expression

Vegetative

Rosette

Root

Supplemental Figure 4. Heatmap showing absolute expression

of selected stomtal regulatory genes in rosettes and roots. Data

was retrieved from publically available datasets (eFP-Browser;

Winter et al., 2007).

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6

CellCell wallDevelopmentDNAProteinRNA regulation of transcription

Signaling receptor kinasesSignaling

Auxin metabolismHormone metabolism

Mitochrondrial ECT/ATP synthesis

PhotosynthesisMajor CHO metabolism

TCA/org. transformation

GlycolysisLipid metabolism

TransportSecondary metabolism

StressRedox regulation

scrm

-D m

ute

CLV3p

ro

FILpro

WUSpro

root ti

ps zone 1

root ti

ps

0 1 0.00010.05

p-value

Supplemental Figure 7. Overrepresentation analysis of transient and permanent stem-cell populations. Columns represent cells expressing the CLV3, FIL, or WUS promoters, respectively. Root tip zone 1 represents all cells up to 150 µm from the root tip of 5 day-old seedlings (Dinneny et al., 2008) and root tip represents cells collected from 130 µm from the root tip, which includes the QC and surrounding stem cells (Sena et al., 2009). Up- and downregulated genes (p≤0.005) were determined through comparison with wild type seed-ling expression. All comparisons are made to wild type seedlings. Dinneny, J.R., Long, T.A., Wang, J.Y., Jung, J.W., Mace, D., Pointer, S., Barron, C., Brady, S.M., Schiefelbein, J., and Benfey, P.N. (2008). Cell identity mediates the response of Arabidopsis roots to abiotic stress. Science 320, 942-945. Sena, G., Wang, X., Liu, H.Y., Hofhuis, H., and Birnbaum, K.D. (2009). Organ regeneration does not require a functional stem cell niche in plants. Nature 457, 1150-1153.


7

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8

Supplemental Table 1. Genes used for production of GFP reporter

constructs.

Gene Identifier Description Fold increase At5g07260 Homeobox related 75-fold At2g19980 TPX-1 related protein 41-fold At1g26600 CLE9 30-fold At2g41190 Amino acid transporter 25-fold At4g28420 Aminotransferase 24-fold At1g33930 AIG-responsive protein related 23-fold At1g52910 Unknown 22-fold At4g31805 POLAR, unknown function 21-fold At5g62210 Embryo-specific protein related 20-fold * At1g12845 Unknown 20-fold At5g07280 EMS1 19-fold At5g13220 JAZ10 15-fold At2g40670 ARR16 Arabidopsis Response

Regulator 14-fold

At3g17640 LRR-family protein 9-fold At4g29740 CKX4 cytokinin oxidase 6-fold * indicates fold-increase was in comparison to spch seedlings.

Supplemental Table 1. Genes used for production of GFP reporter

constructs.

All genes were identified as upregulated in the meristemoid-enriched scrm-D mute genotype as determined by comparison to wild type seedlings (Data Set 4), except where indicated. Gene locus identifiers, gene description, and fold change are listed.


9

Supplemental Table 2. T-DNA insertion lines examined in this studyGene identifier Gene Description T-DNA stockAt1g12845 unknown protein FLAG_472G02At1g26600 CLE9 (CLAVATA3/ESR-RELATED 9); receptor binding SALK_018122At1g33930 avirulence-responsive family protein / avirulence induced gene (AIG1) family protein SALK_060790CAt1g52910 unknown protein SALK_013728At2g19980 allergen V5/Tpx-1-related family protein FLAG_094E06At2g40670 ARR16 (response regulator 16) SAIL_398_C12At2g41190 amino acid transporter family protein SALK_150035At3g17640 leucine-rich repeat family protein SAIL_652_C04At4g29740 CKX4 (CYTOKININ OXIDASE 4) SALK_146112At4g31805 POLAR, unknown function SALK_122914, SALK_122924, SALK_142820At5g13220 JAZ10 SAIL_92_D08At5g62210 embryo-specific protein-related SALK_038225

Supplemental Table 2. T-DNA insertion lines examined in this studyAvailable T-DNA insertion lines for each gene were identified using SIGnAL T-DNA Express Tool (http://signal.salk.edu/cgi-bin/tdnaexpress) and TAIR (www.arabidopsis.org). Gene locus identifiers and gene descriptions are listed.


10

Supplemental Table 3. Genes common to transient and permanent stem-cell populations.

ATH1 array identifier Gene identifier Gene Description

247471_at At5g62230 ERL1__ERECTA-like 1

259083_at At3g04810 ATNEK2_NEK2__NIMA-related kinase 2

256389_at At3g06220 AP2/B3-like transcriptional factor family protein

250642_at At5g07180 ERL2__ERECTA-like 2

266733_at At2g03280 O-fucosyltransferase family protein

256780_at At3g13640 ATRLI1_RLI1__RNAse l inhibitor protein 1

247643_at At5g60450 ARF4__auxin response factor 4

262081_at At1g59540 ZCF125__P-loop containing nucleoside triphosphate hydrolases superfamily protein

257929_at At3g16980 NRPB9A_NRPD9A_NRPE9A__RNA polymerases M/15 Kd subunit

248710_at At5g48480 Lactoylglutathione lyase / glyoxalase I family protein

262802_at At1g20930 CDKB2;2__cyclin-dependent kinase B2;2

259407_at At1g13320 PP2AA3__protein phosphatase 2A subunit A3

259998_at At1g68120 ATBPC3_BPC3__basic pentacysteine 3

262003_at At1g64460 Protein kinase superfamily protein

263239_at At2g16570 ASE1_ATASE_ATASE1__GLN phosphoribosyl pyrophosphate amidotransferase 1

265373_at At2g06510 ATRPA1A_ATRPA70A_RPA1A_RPA70A__replication protein A 1A

257020_at At3g19590 BUB3.1__Transducin/WD40 repeat-like superfamily protein

265087_at At1g03760 Prefoldin chaperone subunit family protein

251470_at At3g59570 Ypt/Rab-GAP domain of gyp1p superfamily protein

Supplemental Table 3. Genes common to transient and permanent stem-cell populations.

List of 19 genes identified as upregulated in scrm-D mute plants (transient stem-cell population) and cells with active CLV3,

FIL, or WUS promoters (permanent stem-cell populations). For all stem-cell populations, significance of upregulation was

determined from comparison to wild type seedlings. ATH1 array, gene locus identifiers, and gene descriptions are given for

each. Gene descriptions were derived from BAR (http://142.150.214.117/welcome.htm).


11

Supplemental Table 4. Primers used for transcript amplification in RT-PCR and qRT-PCRRT-PCRGene Forward Primer Reverse PrimerACTIN2 GCCATCCAAGCTGTTCTCTC GCTCGTAGTCAACAGCAACAAARR16 CACCCTCGAGATGAACAGTTCAGGAGGTTC TGATGACTCCTTCACTTTCCLE9 CACCGTTCGTGTGATTCCTTC CCAGAGGGGACGAGTCTTTTGEPF1 CACCCTCGAGATGAAGTCTCTTCTTCTCCTTG TTGAATTCTAGGGACAGGGTAGGACTTATEPF2 CACCCTCGAGATGACGAAGTTTGTACGCAAGT ACGGCGGAGATTCAATTCAAGERL1 CGCATAACTTGCGGGAATTTG AGTCCTTGTGCAGCTCCAACCERL2 GCTGTGGATAACGAGGCCAAC CATGGTGGGTCTCTCCAAAGGJAZ10 ACGAGTCGTCGATGGAGACAG GGCCGATGTCGGATAGTAAGGMUTE TCGAAGAAGGCAAATGAACGAG GGGTAGTGGCGGCTCCTAAACPOLAR TCTTGGTCCCTCCTACGAAGC GCACGATAGTGCACCCATCTCTMM CACTCACAAGCTGTGGATCGTT GACGGGAATGGACCTGATAACCqRT-PCRGene Forward Primer Reverse PrimerACTIN2 GATGAGGCAGGTCCAGGAATC AACCCCAGCTTTTTAAGCCTTTARR16 CCGATTACTGTATGCCTGGAA TGAGCTCCACTCGCTAAACATCLE9 CACCGTTCGTGTGATTCCTTC CCAGAGGGGACGAGTCTTTTGEPF1 ATGCCGTCTTGTGATGGTTAG TCAAGGGACAGGGTAGGACTTEPF2 TTTGGTCGTTAACTCCATTCG ATCCGGTAAGCTTGATCCTGTERL1 CGCATAACTTGCGGGAATTTG AGTCCTTGTGCAGCTCCAACCERL2 GCTGTGGATAACGAGGCCAAC CATGGTGGGTCTCTCCAAAGGJAZ10 ACGAGTCGTCGATGGAGACAG GGCCGATGTCGGATAGTAAGGMUTE TCGAAGAAGGCAAATGAACGAG GGGTAGTGGCGGCTCCTAAACPOLAR TCTTGGTCCCTCCTACGAAGC GCACGATAGTGCACCCATCTCTMM AGCTGAGGCTCAACGATAACA CCTCAGCTTTCTCCTCATCCTSCZ ACGAGAGATTACGACGAAGCA AACAGGCTTGACATGGTTTTGCDKB2;1 ATGCAAAGGTATGGCATTCTG TGTCTTGGGATCCATCAAGAGCYCA1;1 GAGGCTAAGAAGCGACCTGAT ACGTCTCCGGTACAAGCCTATCYCA2;2 CATCTCCCAAACGAGTCACAT GTTTTCGATGTTGCTTGGTGTCYCA2;3 AAAGACCAGCCTTGGAGGATA ATGACCGCGTCCTTTCTTTATCYCB1;2 TGTACCTCACCGTCAACATCA AAGCAATAAGCAAGGCACTGACYCB1;3 GCTAGAAAACGCACAAGTTGC GCTTTCTTTTGAACCGCTCTTAT5G10890 CCTTCCGAATTGCAAAGAAGG TTGCCTCAGTTCTTCGTTTCG

Supplemental Table 4. Primers used for transcript amplification in RT-PCR and qRT-PCRGene name and sequences for forward and reverse primers are given for each gene amplified. All sequences are in 5’ to 3’ orientation.


12

Supplemental Table 5. Reporter and overexpression constructs produced for this study

Gene identifier Gene description Promoter Genomic coding 35S overexpression GFP protein fusion

base vector pENTR 5'-TOPO base vector pENTR D-TOPO base vector GWB2 base vector R4GWB504

At1g12845 Unknown REY107 REY109 LJP289 LJP257

At1g26600 CLE9 AR114 * KMP119 KMP121 AR116 *

At1g33930 AIG-responsive protein related AR111 AR112 AR137 AR138

At1g52910 Unknown KMP109 REY103 LJP288 LJP276

At2g19980 TPX-1 related protein KMP110 KMP113 LJP251 KMP115

At2g40670 ARR16 Arabidopsis Response Regulator LJP260 LJP261 LJP271 KMP116

At2g41190 Amino acid transporter LJP262 LJP263 LJP290 LJP279

At3g17640 LRR-family protein LJP281 LJP280 KMP118 LJP302

At3g49260 IQD21 calmodulin binding JG109 JG104 KMP150 KMP149

At4g28420 Aminotransferase LJP258 LJP259 LJP270 LJP278

At4g29740 CKX4 cytokinin oxidase RJH21 KUT612 * RJH23 **

At4g31805 POLAR, unknown function KMP112 REY108 LJP256 LJP275

At5g07260 Homeobox-related protein KMP103 KMP105 LJP252 LJP254

At5g07280 EMS1 TK101 * - - TK105 *

At5g13220 JAZ10 LJP168 LJP269 LJP274 KMP117

At5g62210 Embryo-specific protein related LJP264 LJP265 LJP272 LJP282

* base vector pENTR D-TOPO * linker sequence * base vector GWB4 -> promoter:sGFP fusion

** base vector AR132 -> promoter:nls-3x-GFP fusion

Supplemental Table 5. Reporter and overexpression constructs produced for this study

Gene identifier, gene description, and name of each construct are indicated. Base vectors for each construct are given.

See Materials and Methods for base vector information.


13

Supplemental Table 6. Primers used for production of constructs used in this study.

Gene identifier Promoter Forward Promoter Reverse Coding Forward Coding Reverse

At1g12845 (-639) CCTCATCATCATCAAATTC (-1) GGGTGTTTCTTTTTCTTCTG ATGGCTCTGTTTTCTAGCGT ATAGTGTAGAAACCTAACAAG

At1g26600 (-974) ATCAGAACCTGAGAACTATAG (-1) TGTTTTGGTTTCCAAGAGAG ATGACGATGACTCACCTGAATCGTCTGATCTTGATCTCTC GTTGTGAAGCGGGTTTGGACCAGAGGGGACG

At1g33930 (-1134) CTCTCTTGAATCGTTTCATA (-1) CTGAAAAAGAAAAAACAAAT ATGGCTGAAGATCAGAACACAACTGACCG TAACTCTTTTTCCATCCACCTCTCT

At1g52910 (-1600) CACACTACACACAGCTTTTTC (-1) TGCGATTATTCTGAGAGAGC ATGGCTTCAAAGCTCGTGAT GTAATGTGGGGTTGGGTAAC

At2g19980 (-1844) TGATTGGATTTTATTATACTG (-1) TTTCTCTCTTTTGTTTTTGTTG ATGTCTTTTTCAGGTTATAG GTACGGACGAGTATTAGC

At2g40670 (-1391) TAAACTCAGTTGATACTTC (-1) GATCTCCTTTGTTCCTTAAA ATGAACAGTTCAGGAGGTTC GCTTCTGCAGTTCATGAGAT

At2g41190 (-1511) ATTGTGATAACTTCCTTC (-1) ATTTCGAACAAACCCAAGTA ATGGAGGACAAGAACAATG CTGATAGTTCCTAATGATCT

At3g17640 (-639) CTGCCTGAACGGATTTTATT (-1) GTTTTTGTTTTTGAGAGTTG ATGAGCTACACTTTCTTTC ACCAGTGGAGCTGTGATTA

At4g28420 (-840) AAACTCAGCTTCATTCTCGG (-1) TGTGAATATATTCGTTGGG ATGAACCACAACAGCAACC AGAAATAAGAATCATCCAAAT

At4g29740 (-2074) TGTTGCTAAGCTCTTCTTCTTC (-1) AATCTAACAAACGGGTAGGTTAA - -

At4g31805 (-641) ACGTTTTTAGTGAATGTATT (-1) TGAAAACCAAGAGAAGACA ATGGAAGGCTCGCGGCG TCTAGAAAAACTGGTGCGGT

At5g07260 (-2124) AGTGATCTCTCTCTTCTCTG (-1) ATCTGACCTTTTCATGTGTT ATGTATCATCATCTTCAAACG ACGGTGGACTGGAAGAGCAT

At5g07280 (-1740) CAGAGAGAACCAATGCAACTC (-1) GTTCTTTTAGAGAAGGAGGAAGAAAACTCGGAG - -

At5g13220 (-2393) GAGTTTGGTTTTGTATTCC (-1) CTTCTTTGATCTTATTAGAA ATGTCGAAAGCTACCATAG GGCCGATGTCGGATAGTAA

At5g62210 (-978) TGATTGGATAAAATTATTT (-1) TGTTGTCTTCTTGAAAAGA ATGGAGTGCTCTCTCTCAT AACAACCACAGCCGCAAACG

Supplemental Table 6. Primers used for production of constructs used in this study.

Gene identifiers, gene descriptions, and respective sequences for promoter and coding region amplification are

given. All sequences are in 5’ to 3’ orientation. Numbers in parentheses for promoter amplification indicate

primer location relative to the translational start site.


14

Documents

Pillitteri Fig S1 - Plant Cell · TMM MUTE ERL1 ERL2 EPF1 EPF2 CLE9 ARR16 JAZ10 POLAR ACT2 WT spch scrm-D mute scrm-D gDNA (-) Supplemental Data. Pillitteri et al. (2011). 10.1105/tpc.111.088583