Elongator is required for root stem cell maintenance by ... · 11/6/2018 · 102 and ELP2 functioning as scaffolds for complex assembly, ELP3 acting as the catalytic 103 subunit,

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Short title: Elongator regulates SHR transcription 1

Corresponding author details: 2

Dr. Qian Chen 3

State Key Laboratory of Crop Biology 4

College of Agronomy 5

Shandong Agricultural University 6

Tai'an, 271018. 7

Shandong Province, China 8

E-mail: [email protected] 9

Tel: +86-538-8241758 10

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Title 12

Elongator is required for root stem cell maintenance by regulating 13

SHORT ROOT transcription 14

Authors: Linlin Qi1,2,4

, Xiaoyue Zhang2,3,4

, Huawei Zhai

2,3,4, Jian Liu

1,4, Fangming 15

Wu2, Chuanyou Li

2,4* and Qian Chen

1* 16

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1State Key Laboratory of Crop Biology, College of Agronomy, Shandong 18

Agricultural University, Taian, Shandong 271018, China 19

2University of Chinese Academy of Sciences, Beijing 100049, China 20

3State Key Laboratory of Plant Genomics, National Centre for Plant Gene Research, 21

Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, 22

Beijing 100101, China 23

Plant Physiology Preview. Published on November 6, 2018, as DOI:10.1104/pp.18.00534

Copyright 2018 by the American Society of Plant Biologists

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One-sentence summary: 24

Elongator subunits associate with the pre-mRNA of SHORT ROOT and recruit 25

RNAPII to the SHR gene body, and thereby contribute to root stem cell maintenance 26

and radial patterning. 27

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Footnotes: 29

Author contributions 30

L.Q. and X.Z carried out genetic assays and genotyped the mutants; X.Z. and H.Z. 31

undertook the confocal microscopy; J.L. made the constructs and prepared the 32

transgenic plants; F.W. carried out the biochemical assays. X.Z. and Q.C. designed the 33

project and drafted the manuscript with contributions of all the authors. C.L. and Q.C. 34

supervised and complemented the writing. 35

4These authors contribute equally to this work. 36

Funding: 37

This work was supported by the National Basic Research Program 38

of China (2015CB942900 and 2013CB967301); the Tai-Shan Scholar Program from 39

the Shandong Province, the State Key Laboratory of Plant Genomics of China, and 40

the State Key Lab of Crop Biology of China; the National Natural Science Foundation 41

of China (31320103910). 42

Present address: 43



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Q.L.: State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary 44

Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China 45

*Correspondence: Chuanyou Li ([email protected]), Qian Chen 46

([email protected]) 47

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Abstract 49

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SHORTROOT (SHR) is essential for stem cell maintenance and radial patterning in 51

Arabidopsis (Arabidopsis thaliana) roots, but how its expression is regulated is 52

unknown. Here, we report that the Elongator complex, which consists of six subunits 53

(Elongator1–6), regulates the transcription of SHR. Depletion of Elongator drastically 54

reduced SHR expression and led to defective root stem cell maintenance and radial 55

patterning. The importance of the nuclear localization of Elongator for its functioning, 56

together with the insensitivity of the elp1 mutant to the transcription elongation 57

inhibitor 6-azauracil, and the direct interaction of the ELP4 subunit with the 58

C-terminal domain of RNA polymerase II (RNAPII CTD), support the notion that 59

Elongator plays important roles in transcription elongation. Indeed, we found that 60

ELP3 associates with the pre-mRNA of SHR and that mutation of Elongator reduces 61

the enrichment of RNAPII on the SHR gene body. Moreover, Elongator interacted in 62

vivo with SUPPRESSOR OF Ty4 (SPT4), a well-established transcription elongation 63

factor that is recruited to the SHR locus. Together, these results demonstrate that 64

Elongator acts in concert with SPT4 to regulate the transcription of SHR. 65

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Introduction 67

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Root growth in higher plants relies on a group of pluripotent, mitotically active stem 69

cells residing in the root apical meristem (RAM). In the RAM of the model plant 70

Arabidopsis (Arabidopsis thaliana), the mitotically less active quiescent center (QC) 71

cells, together with their surrounding stem cells, constitute the root stem cell niche 72

(SCN), which continues to provide cells for all root tissues (van den Berg et al., 1995). 73

Pioneering studies have identified several key regulators that help determine the 74

specification and functioning of the SCN. Among these, the GAI, RGA, and SCR 75

(GRAS) family transcription factors SHORTROOT (SHR) and SCARECROW (SCR) 76

provide positional information along the radial axis, whereas the plant hormone auxin, 77

together with its downstream components, the PLETHORA (PLT) class of 78

transcription factors, provide longitudinal information (Di Laurenzio et al., 1996; 79

Helariutta et al., 2000; Aida et al., 2004; Aichinger et al., 2012). 80

In addition to regulating the positional specification of the QC, SHR also 81

controls the formative division of the cortex/endodermis initial (CEI) stem cell and its 82

immediate daughter cell (CEID), which generates the separate endodermis and cortex 83

cell layers constituting root ground tissue (van den Berg et al., 1995). Interestingly, 84

SHR is transcriptionally expressed in the stele, and its encoded protein moves into the 85

outer adjacent cell layer, where its partner SCR sequesters SHR to the nucleus by 86

forming the SHR-SCR complex (Nakajima et al., 2001; Cui et al., 2007). Recent 87

efforts have successfully identified important transcriptional targets of the SHR-SCR 88



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complex. Among these are a group of so-called BIRD family genes encoding zinc 89

finger proteins (Levesque et al., 2006; Welch et al., 2007; Long et al., 2015) and the 90

cell-cycle gene CYCLIN D6;1 (CYCD6;1) (Sozzani et al., 2010). The spatiotemporal 91

activation of CYCD6;1 is controlled by a bistable switch involving SHR, SCR, and the 92

cell differentiation factor RETINOBLASTOMA-RELATED (RBR), which is also 93

regulated by the formation of a dynamic MED31-SCR-SHR ternary complex 94

(Cruz-Ramírez et al., 2012; Zhang et al., 2018). Despite these advances, how the 95

master regulator gene SHR itself is regulated remains largely unknown. 96

In eukaryotic cells, protein-coding genes are transcribed by RNA polymerase II 97

(RNAPII). The multifunctional protein complex, Elongator, was first identified as an 98

interactor of hyperphosphorylated (elongating) RNAPII in yeast and was later purified 99

from human and Arabidopsis cells (Otero et al., 1999; Hawkes et al., 2002; Nelissen 100

et al., 2010). Elongator consists of six subunits, designated ELP1 to ELP6, with ELP1 101

and ELP2 functioning as scaffolds for complex assembly, ELP3 acting as the catalytic 102

subunit, and ELP4-6 forming a subcomplex important for substrate recognition 103

(Versees et al., 2010; Glatt et al., 2012; Woloszynska et al., 2016). In yeast, the loss of 104

Elongator subunits leads to altered sensitivity to stresses including salt, caffeine, 105

temperature, and DNA damaging agents (Otero et al., 1999; Krogan and Greenblatt, 106

2001; Esberg et al., 2006). Since Elongator was copurified with elongating RNAPII 107

and the ELP3 subunit showed histone acetylation activity, it was initially proposed 108

that Elongator mainly functions as a transcription elongation factor, a process that 109

occurs in the nucleus (Otero et al., 1999; Wittschieben et al., 1999; Winkler et al., 110



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2002). Shortly thereafter, this proposition was questioned, as several studies show that 111

yeast Elongator has diverse functions related to its transfer (t)RNA modification 112

activity that take place in the cytoplasm (Huang, 2005; Esberg et al., 2006; Li et al., 113

2009; Chen et al., 2011; Bauer et al., 2012; Fernández-Vázquez et al., 2013). 114

The physiological functions of Elongator in mammals are exemplified by the 115

finding that impaired Elongator activity in human is correlated with the neurological 116

disorder familial dysautonomia (Anderson et al., 2001) and that mutations in 117

Elongator subunits are embryotic lethal in mice (Chen et al., 2009). Like its yeast 118

counterpart, human Elongator also has lysine acetyltransferase (KAT) activity. Among 119

the major substrates for the KAT activity of human Elongator are Histone H3 and 120

α-tubulin, reflecting the distinct functions of Elongator in the nucleus and cytoplasm. 121

While, in the nucleus, acetylation of Histone H3 is linked to the function of Elongator 122

in transcription (Svejstrup, 2007), cytoplasmic acetylation of α-tubulin by Elongator 123

underlies the migration and maturation of neurons (Creppe et al., 2009). 124

Genetic studies have demonstrated that Elongator plays an important role in 125

regulating multiple aspects of plant development and adaptive responses to biotic and 126

abiotic stresses (Nelissen et al., 2005; Zhou et al., 2009; Nelissen et al., 2010; Wang et 127

al., 2013; Jia et al., 2015). Recent studies reveal the role of plant Elongator in 128

regulating microRNA biogenesis and tRNA modification (Fang et al., 2015; Leitner 129

et al., 2015). 130

Here, we report the action mechanism of plant Elongator in regulating root SCN 131

and radial patterning. We show that the root developmental defects of Elongator 132



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mutants are largely related to drastically reduced SHR expression. We provide 133

evidence that Elongator acts as a transcription regulator of SHR. 134

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Results 136

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Elongator is required for SCN maintenance and general root growth 138

To systematically evaluate the role of Elongator in regulating root growth, we 139

investigated mutants of all six Elongator subunits (elp1 to elp6, see Materials and 140

Methods) and several double mutants including elp1 elp2, elp1 elp4, elp1 elp6, elp2 141

elp4, elp2 elp6, and elp4 elp6. Each of the single mutants exhibited similar reductions 142

in root growth, and none of the investigated double mutant lines showed additive 143

effects (Supplemental Fig. S1A), implying that each subunit is essential for the 144

functioning of Elongator and that Elongator acts as an integral complex that regulates 145

root growth. Therefore, we used elp1 as a representative mutant for detailed 146

phenotypic analyses. 147

Cytological observations revealed that both cell division and cell elongation 148

were reduced in elp1 (Supplemental Fig. S1, B-H). In a Lugol's iodine starch staining 149

assay of wild-type (WT) roots expressing the QC-specific marker QC25, one layer of 150

columella stem cells (CSCs) without starch staining was visible between the QC and 151

the columella cell layers, hinting at a well-organized and functional SCN (Fig. 1A). 152

By contrast, in elp1 root tips, QC25 expression was weak in the QC, but its expression 153

pattern expanded downward and merged with that of starch staining, and the CSCs 154



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could not be clearly discerned (Fig. 1B), suggesting the loss of QC cell identity and 155

CSC differentiation. Consistently, in RNA in situ hybridization assays of WT roots, 156

WUSCHEL-RELATED HOMEOBOX 5 (WOX5) was specifically expressed in the QC, 157

but its expression pattern was diffuse and merged with neighboring cells in elp1 roots 158

(Fig. 1, C and D). These results, together with previous observations of the elp2 159

mutant (Jia et al., 2015), indicate that Elongator is required for root SCN maintenance 160

and general root growth. 161

162

Elongator regulates radial patterning in roots through the SHR pathway 163

To investigate the genetic relationship between Elongator and the SHR pathway, we 164

generated an elp1 shr-2 double mutant line. At 5 days after germination (DAG), 165

general root growth and the meristem cell number of the double mutant were similar 166

to those of shr-2 (Fig. 1, E and F), indicating that elp1 and shr-2 do not have additive 167

effects on root growth. These results support the notion that Elongator acts genetically 168

in the SHR pathway to regulate root growth. 169

In parallel experiments, the elp1 plt1-1 plt2-4 triple mutant line appeared to show 170

an additive effect compared with its parental lines, elp1 and plt1-1 plt2-4 171

(Supplemental Fig. S2, A-F). Consistently, the elp1 mutation had only a minor effect 172

(if any) on PLT1 and PLT2 expression (Supplemental Fig. S2, G-J). These results 173

support the notion that Elongator acts genetically in parallel with the PLT pathway. 174

In addition to having a defective SCN, shr mutants also exhibit irregular radial 175

patterning and reduced stele width (Levesque et al., 2006). Hence, we examined 176



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whether elp1 shows similar phenotypes. The anatomical organization showed that the 177

cortex and endodermal cellular patterns of elp1 were disordered compared with WT 178

(Supplemental Fig. S3, A and B); CEI/CEID did not divide correctly and formed a 179

single cell layer in shr-2, and elp1 shr-2 showed similar phenotypes with shr-2 180

(Supplemental Fig. S3, C and D). Using the cortex-specific marker pCO2:H2B-YFP 181

and the endodermis-specific marker pSCR:GFP-SCR, we clearly distinguished these 182

well-organized cell layers in WT roots (Fig. 1, G and I), whereas irregular patterning 183

in certain regions of the cortex and/or endodermis cell layers was frequently observed 184

in elp1 roots (Fig. 1, H and J). Consistently, the expression levels of CO2 and SCR 185

were lower in elp1 than in the WT (Fig. 1, G-N). In elp1, the expression of cortex and 186

endodermal marker J0571 was also disturbed, and almost undetectable in some 187

ground tissue cells, which demonstrates that these cells in elp1 lost their cell identity 188

(Supplemental Fig. S3, E and F). Moreover, the stele width at the transition zone was 189

also significantly reduced in elp1 compared with the WT (Fig. 1, G-O). Together, the 190

phenotypic similarity between elp1 and shr strengthens the idea that Elongator acts in 191

the SHR pathway to regulate radial patterning in roots. 192

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Depletion of Elongator impairs the expression of SHR and its target genes 194

Using the SHR promoter fusion line pSHR:erGFP (for green fluorescent protein) and 195

the SHR protein fusion line pSHR:SHR-GFP, we found that SHR expression levels 196

were drastically reduced in elp1 compared with the WT (Fig. 2, A-D). This 197

observation was confirmed by RNA in situ hybridization (Fig. 2, E and F) and 198



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reverse-transcription quantitative polymerase chain reaction (RT-qPCR) assays (Fig. 199

2G). Not surprisingly, as revealed by RT-qPCR, the expression levels of several SHR 200

transcriptional targets, including SCR, BR6ox2, CYCD6;1, MGP, NUTCRACKER 201

(NUC), RECEPTOR-LIKE KINASE (RLK), and SNEEZY/SLEEPY 2 (SNE), were also 202

substantially reduced in elp1 compared with the WT (Fig. 2G). We then investigated 203

whether the elp1 mutation impairs the expression of the cell-cycle gene CYCD6;1 in 204

the CEI/CEID. Previous studies have elegantly demonstrated that the spatiotemporal 205

activation of CYCD6;1 coincides with the formative division of CEI/CEID and that 206

this process is strictly controlled by SHR and the related transcription factor SCR 207

(Sozzani et al., 2010; Cruz-Ramírez et al., 2012). As expected, the spatiotemporal 208

expression of CYCD6;1 in the CEI/CEID was largely disrupted in the elp1 mutant 209

(Fig. 2, H and I). Together, these results indicate that the depletion of Elongator 210

impairs the expression of SHR and its target genes. 211

To visualize the expression pattern of the Elongator subunit ELP1, we fused the 212

ELP1 promoter with the glucuronidase (GUS) reporter and generated pELP1:GUS 213

transgenic plants. GUS staining revealed that, like SHR, ELP1 was highly expressed 214

in the stele of the root tip (Fig. 2J). This observation strengthens the notion that 215

Elongator regulates root development through the SHR pathway. 216

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Nuclear localization of Elongator is important for its function in regulating root 218

development 219



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To determine the subcellular localization of Elongator, we introduced the 220

pELP3:ELP3-GFP fusion construct into elp3 plants. The functionality of the 221

ELP3-GFP fusion protein was verified by its ability to rescue the root growth defects 222

of the elp3 mutant (Supplemental Fig. S4A). Confocal microscopy of 223

elp3;pELP3:ELP3-GFP plants indicated that, in stele cells of the meristem region, 224

ELP3-GFP was predominantly localized to the cytoplasm and, to a lesser extent, the 225

nucleus (Fig. 3, A and B). Interestingly, we observed more obvious nuclear 226

localization in columella cells and epidermis cells at the elongation zone in the 227

previously reported line, p35S:GFP-ELP3 (Supplemental Fig. S4B). 228

We then employed a cell fractionation approach to determine the subcellular 229

localization of endogenous ELP1 and ELP3. For these experiments, protein extracts 230

of WT seedlings were fractionated and probed with antibodies that specifically 231

recognize endogenous ELP1 or ELP3 protein (Supplemental Fig. S5, A and B). 232

Histone H3 was exclusively detected in the nuclear compartment, whereas 233

phosphoenolpyruvate carboxylase (PEPC) was only detected in the cytoplasmic 234

compartment, validating our approach. Consistent with the above cytological 235

observations, endogenous ELP1 and ELP3 were clearly detected in both the 236

cytoplasmic and nuclear fractions (Fig. 3C). We obtained similar results from cell 237

fractionation experiments using elp3;p35S:ELP3-myc transgenic plants, in which the 238

root growth defects of the elp3 mutant had been rescued (Supplemental Fig. S5C). 239

These results help confirm the finding that Arabidopsis Elongator is located in both 240

the cytoplasm and the nucleus. 241



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To determine if the nuclear localization of ELP3 is critical for its function, we 242

artificially confined ELP3 to the nuclear compartment and investigated whether it 243

would still retain its function. For these experiments, ELP3-GFP was fused with the 244

efficient nuclear localization sequence SV40 NLS (van der Krol and Chua, 1991) to 245

generate the p35S:NLS-ELP3-GFP construct. Analysis of the resulting transgenic 246

plants indicated that the NLS-ELP3-GFP fusion protein was successfully translocated 247

into the nucleus and, more importantly, the nuclear-localized NLS-ELP3-GFP fusion 248

protein was functional, as it fully rescued the root growth defects of elp3 (Fig. 3, D-F). 249

Histone H3 lysine 14 acetylation level is slightly reduced in elp1 and elp3, which is 250

consistent with the nuclear localization and the reported histone modification function 251

of the Elongator complex (Supplemental Fig. S5D). These results support the notion 252

that the nuclear localization of Elongator is important for its function in regulating 253

root development. 254

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Elongator functions as a transcription elongation factor to regulate SHR 256

transcription 257

Our finding that the nuclear localization of plant Elongator is important for its 258

biological function suggested that Elongator might act as a transcription elongation 259

factor involved in RNAPII-dependent transcription. To investigate this notion, we 260

first examined whether the Elongator subunits interact with the conserved C-terminal 261

domain (CTD) of RNAPII, an interaction platform between RNAPII and other 262

proteins involved in transcription. 263



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RNAPII CTD interacted with ELP4 and ELP5 in yeast two-hybrid (Y2H) assays 264

(Fig. 4A). To determine whether ELP4 interacts with CTD in planta, we conducted 265

firefly luciferase (LUC) complementation imaging (LCI) assays in Nicotiana 266

benthamiana leaves. In these experiments, ELP4 was fused to the N-terminal half of 267

LUC (nLUC) to produce ELP4-nLUC, whereas CTD was fused to the C-terminal half 268

of LUC (cLUC) to produce cLUC-CTD. N. benthamiana cells co-expressing 269

ELP4-nLUC and cLUC-CTD displayed strong luminescence signals, whereas those 270

co-expressing nLUC and cLUC-CTD or ELP4-nLUC and cLUC displayed no signal 271

(Fig. 4, B and C), confirming that the ELP4–CTD interaction occurs in vivo. 272

We then examined the response of elp1 to 6-Azauracil (6-AU), an inhibitor of 273

enzymes involved in purine and pyrimidine biosynthesis. In yeast, 6-AU is a widely 274

used inhibitor of transcription elongation, as it alters nucleotide pool levels in vivo 275

(Exinger and Lacroute, 1992). Strikingly, elp1 was more resistant to 6-AU-induced 276

root growth inhibition than the WT (Fig. 4D), providing another line of evidence that 277

Elongator functions as a transcription elongation factor in Arabidopsis. 278

Next, we investigated whether Elongator participates in RNAPII-dependent 279

transcription elongation of SHR using chromatin immunoprecipitation (ChIP)-qPCR 280

assays. In WT plants, CTD was highly enriched on both the transcription start site 281

(TSS) and gene body of SHR (Fig. 4E). In the elp1 mutant, however, CTD levels on 282

the SHR locus were significantly reduced (Fig. 4E), revealing that Elongator is 283

important for the recruitment of RNAPII to the SHR locus during SHR transcription. 284



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This line also demonstrates that Elongator is mainly involved in transcription 285

elongation, rather than transcription initiation. 286

We then performed RNA-immunoprecipitation (RNA-IP) assays to investigate 287

whether Elongator associates with the pre-mRNA of SHR. Specifically, we used the 288

GFP antibody to immunoprecipitate ELP3-GFP from extracts of the 289

elp3;p35S:NLS-ELP3-GFP transgenic line. The resulting ELP3-GFP 290

immunoprecipitates were then reverse transcribed into cDNAs and subjected to 291

RT-PCR with primers specific for SHR, SCR, PLT2, or ACTIN7 (ACT7). Pre-mRNA 292

of SHR was detected in immunoprecipitates from the elp3;p35S:NLS-ELP3-GFP line 293

but not from those of the WT (Fig. 4F), confirming that ELP3 indeed associates with 294

SHR pre-mRNA. As a control, SCR, PLT2 and ACT7 pre-RNAs were not detected in 295

the same ELP3-GFP immunoprecipitates (Fig. 4F), suggesting that ELP3 specifically 296

associates with the pre-mRNA of SHR. Together, these results led us to conclude that 297

Elongator regulates the transcription of SHR through associating with its pre-mRNA. 298

ELP1 associates with the transcription elongation factor SPT4, which is 299

recruited to the SHR locus 300

Our results support the notion that Elongator regulates the transcription elongation of 301

SHR through associating with the pre-mRNA of SHR. Intriguingly, however, we 302

failed to detect Elongator enrichment on the SHR locus in the ChIP experiments. We 303

speculated that Elongator might act in concert with other known transcription 304

elongation factors to regulate the elongation of SHR transcript. Indeed, ELP1 and 305

ELP3 were recently affinity copurified with SUPPRESSOR OF Ty4 (SPT4) and 306



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several other conserved transcription elongation factors in eukaryotic cells (Durr et al., 307

2014; Antosz et al., 2017). In Arabidopsis, SPT4 is encoded by two redundant genes, 308

designated SPT4-1 and SPT4-2 (Durr et al., 2014). In a coimmunoprecipitation (Co-IP) 309

assay using p35S:SPT4-2-GFP plants and anti-ELP1 antibodies, SPT4-2-GFP pulled 310

down native ELP1, indicating that ELP1 associates with SPT4-2 in vivo (Fig. 5A). 311

The in vivo association of ELP1 with SPT4-2 was further confirmed in LCI assays: N. 312

benthamiana cells co-expressing ELP1-nLUC and cLUC-SPT4-2 displayed strong 313

luminescence signals (Fig. 5, B and C). 314

To demonstrate that SPT4-2 plays a role in root development, we generated 315

SPT4-RNAi plants in which the expression of both SPT4-2 and SPT4-1 was knocked 316

down by RNA interference (RNAi) (Supplemental Fig. S6). Like elp1 and the other 317

elp mutants (Supplemental Fig. S1), the SPT4-RNAi plants also showed reduced root 318

growth (Fig. 5D) and irregular patterning in certain regions of the cortex and/or 319

endodermis cell layers (Supplemental Fig. S6, C-E). These results are consistent with 320

a previous observation, and they suggest that the interplay between Elongator and 321

SPT4/SPT5 might help regulate root development (Durr et al., 2014; Van Lijsebettens 322

et al., 2014; Woloszynska et al., 2016; Antosz et al., 2017). Furthermore, like the elp1 323

mutant, SPT4-RNAi plants were less sensitive to 6-AU-induced root growth inhibition 324

than WT plants (Fig. 5D), implying that the function of SPT4-2 in regulating root 325

growth is related to transcription elongation. Indeed, the ChIP-qPCR assays revealed 326

that SPT4-2 was enriched on the SHR locus and importantly, the levels of SPT4-2 on 327

the SHR gene body regions were higher than those on the SHR promoter region (Fig. 328



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5E), suggesting that SPT4 is mainly involved in SHR transcription elongation. Taken 329

together, our results support the notion that Elongator acts in concert with SPT4/SPT5 330

to regulate the transcription of SHR, thereby regulating root development. 331

332

Discussion 333

334

Elongator is required for root SCN maintenance and radial patterning through 335

regulating SHR gene expression 336

In addition to the reduced primary root growth and defective SCN reported previously 337

(Jia et al., 2015), we observed irregular radial patterning and reduced stele width in 338

Elongator single and double mutants, indicating that Elongator functions as an 339

integral complex in the regulation of root development. Several lines of evidence 340

indicate that the developmental defects in roots of the Elongator mutants are largely 341

due to an impaired SHR pathway. Indeed, the elp1 root phenotypes resemble those of 342

shr (Fig. 1) and coincide with the similar developmental gene expression patterns in 343

the primary root, with the highest expression in the stele tissue of root tips. Elongator 344

acts upstream of SHR and independently of the PLT pathway, as the expression levels 345

of SHR and its target genes SCR and CYCD6;1 were drastically reduced in elp1 (Fig. 346

2, A-I), whereas the expression of PLT1 and PLT2 was less affected in this mutant 347

(Supplemental Fig. S2). Thus, Elongator regulates root development mainly through 348

its impact on the SHR pathway. In contrast to our knowledge about SHR target genes 349

and interacting proteins, little had been known about how SHR itself is regulated, 350



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except that SHR expression is not regulated by PLT1, PTL2, SCR, or SHR itself 351

(Helariutta et al., 2000; Aida et al., 2004). Our results indicate that Elongator is a 352

regulator of SHR expression. The developmental and external stimuli that regulate the 353

expression of the genes encoding the six Elongator subunits, such as hormones and 354

temperature (Woloszynska et al., 2016), might affect the production of Elongator 355

subunits, thus affecting the assembly and accumulation of this crucial complex and 356

thereby controlling (to some extent) SHR gene expression. Elongator might serve as 357

an interface between these stimuli and SHR, and the regulation of the SHR gene at the 358

transcription elongation stage might render its expression more flexible and 359

responsive, which is important for the spatial and temporal control of root 360

development. 361

362

Elongator regulates transcription of SHR in concert with SPT4/SPT5 363

The role of Elongator as a transcription elongation factor is highly controversial in 364

yeast and humans (Glatt et al., 2012) and was recently suggested in plants 365

(Woloszynska et al., 2016). Here, using various experimental approaches, we provide 366

direct evidence for the transcription elongation activity of Elongator. Using confocal 367

microscopy analysis of various transgenic lines combined with cell fractionation, we 368

showed that the Elongator subunits are partially localized to the nucleus, although 369

major proportions of these subunits are localized to the cytoplasm (Fig. 3). Moreover, 370

the nuclear localization of these subunits is important for their biological function, as 371

artificially nucleus-localized ELP3 was still able to complement the elp3 mutant 372



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phenotype (Fig. 3, D-F); a similar experiment was performed in yeast, but with 373

contrasting results (Rahl et al., 2005). Our protein purification assays revealed a direct 374

interaction of the ELP4 subunit with RNAPII CTD (Fig. 4, A-C) and the association 375

of ELP1 with the well-established transcription elongation factor SPT4/SPT5 (Fig. 5, 376

A-C), indicating that Elongator is involved in transcription elongation. However, we 377

failed to detect an interaction between ELP4 and RNAPII in a Co-IP assay. This result 378

confirms a previous report showing interactions between the different subunits of 379

Elongator but no interactions between Elongator subunits and RNAPII using TAP-MS 380

(tandem affinity purification/mass spectroscopy) (Nelissen et al., 2010), but disagrees 381

with a more recent report showing co-purification of the two largest subunits of 382

RNAPII with Elongator subunits ELP1 and ELP3 using SPT4 as bait (Durr et al., 383

2014). Hence, the association of Elongator with RNAPII in vivo might be transient 384

and dynamic (Van Lijsebettens et al., 2014). 385

Yeast mutants defective in transcription elongation exhibit an altered sensitivity 386

to 6-AU (Nakanishi et al., 1995; Wu et al., 2003). We found that both elp1 and 387

SPT4-RNAi plants were highly resistant to 6-AU treatment (Fig. 4D and 5D), 388

suggesting that Elongator plays a similar role in transcription elongation to that of 389

SPT4/SPT5. Finally, we detected a significant reduction in the enrichment of RNAPII 390

CTD on the SHR locus in elp1 compared with the WT (Fig. 4E), as well as an 391

association of ELP3 with SHR mRNA (Fig. 4F). Moreover, its associated protein, 392

SPT4, was also recruited to SHR chromatin (Fig. 5E). These findings indicate that 393

both Elongator and SPT4/SPT5 are directly involved in the transcription regulation of 394



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SHR. However, we failed to detect significant enrichment of Elongator on the SHR 395

region via ChIP, possibly due to a lack of DNA-binding activity and/or the dynamic 396

properties of its interaction with other proteins. No ChIP data are currently available 397

for the enrichment of Elongator on transcriptional regulatory regions in Arabidopsis. 398

Hence, we propose a model in which Elongator acts in concert with SPT4/SPT5 to 399

maintain the transcription of SHR. In this model, Elongator directly interacts with the 400

RNAPII CTD through the ELP4 subunit and associates with SHR mRNA, whereas 401

SPT4/SPT5 associates with RNAPII and Elongator, as well as the chromosomal 402

region harboring SHR (Fig. 5F). 403

Transcription elongation is a tightly controlled, dynamic process that can be 404

divided into three distinct stages: promoter escape, promoter-proximal pausing, and 405

productive elongation. Based on their activities, transcription elongation factors can 406

be categorized as positive or negative. In yeast, mutations of positive transcription 407

elongation factors are often associated with hypersensitivity to 6-AU, whereas the 408

disruption of negative transcription elongation factors renders the cells less sensitive 409

to 6-AU (Wu et al., 2003). We demonstrated that Elongator associates with 410

SPT4/SPT5 (Fig. 5, A-C) and that both elp1 and SPT4-RNAi are highly insensitive to 411

6-AU (Fig. 4D and 5D), suggesting that Elongator and SPT4/SPT5 are negative 412

transcription elongation factors, as in yeast. Indeed, human SPT4/SPT5, known as 413

DSIF (5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) sensitivity-inducing 414

factor), is a negative transcription elongation factor that contributes to 415

promoter-proximal pausing (Yamaguchi et al., 2013). Transcriptional pausing is also 416



20

20

thought to participate in mRNA synthesis, possibly through the formation of a 417

“preactivated” state (Yamaguchi et al., 2013). Hence, Elongator and SPT4/SPT5 418

might play an (as yet) unknown role in promoter-proximal pausing in Arabidopsis. 419

Moreover, our ChIP-qPCR results indicated that CTD enrichment on the SHR 420

promoter (around TSS region), which also associated by SPT4, was reduced in the 421

elp1 mutant (Fig. 4E and 5E). These imply that the ELP-SPT4-Pol II complex not 422

only regulates SHR transcription elongation, but also transcription initiation, which is 423

consistent with the abolished expression of pSHR:erGFP (Fig. 2 A and B) and also 424

supports that Elongator and SPT4/SPT5 may function in promoter-proximal pausing. 425

426

Functional diversification of Elongator in eukaryotes 427

In yeast, tRNA modification appears to be the direct, unequivocal biochemical 428

function of Elongator, because its cytoplasmic localization (Rahl et al., 2005) 429

excludes the possibility of this complex being a transcription elongation factor. In 430

addition, overexpressing two related tRNA species rescued almost all of the reported 431

phenotypes of yeast Elongator mutants, including reduced H3K14Ac levels, 432

suggesting that even histone acetylation might be an indirect effect of Elongator 433

activity (Esberg et al., 2006). Various biological processes are modulated through 434

Elongator tRNA modifications, such as telomeric gene silencing (Chen et al., 2011), 435

cell-cycle control (Bauer et al., 2012), and oxidative stress responses 436

(Fernández-Vázquez et al., 2013). However, in animal cells, Elongator is partially 437

localized to the nucleus (Hawkes et al., 2002; Creppe et al., 2009), and its role in 438



21

21

transcription elongation is supported by the enrichment of Elongator subunits on 439

certain genes related to cell migration. Here, we provided direct evidence that 440

Elongator acts as a transcription regulator in Arabidopsis. Since Elongator was first 441

copurified with elongating RNAPII from total cell extracts in yeast (Otero et al., 442

1999), and their interaction was further confirmed in human cells (Hawkes et al., 2002) 443

and here in Arabidopsis, it is reasonable to suspect that the association of Elongator 444

with RNAPII in yeast is not merely a coincidence. Thus, we propose that both tRNA 445

modification and RNAPII association are two functions of Elongator. However, the 446

cytoplasmic localization of this complex in yeast precludes it from being a 447

transcriptional regulator, and therefore the tRNA modification activity of Elongator 448

contributes the most to its function. By contrast, in human and plant cells, the 449

acquisition of a partial nuclear localization for this complex might have caused it to 450

develop a capacity for transcriptional regulation. In animals, the cytoplasm-localized 451

Elongator evolved various other activities, such as acetylation of α-tubulin in the 452

mouse cortex (Creppe et al., 2009). In plants, Elongator also plays roles in the 453

cytoplasm, namely, its tRNA modification activity is conserved in Arabidopsis 454

(Versees et al., 2010), but how this localization contributes to its biological function is 455

still uncertain, although auxin responses depend on Elongator tRNA activity (Leitner 456

et al., 2015). 457

Transcriptional regulation is a major, delicate regulatory mechanism involving 458

numerous proteins. The key players in postembryonic root development in plants, 459

such as PLT1, PLT2, SHR, SCR, and WOX5, are all transcription factors (Aichinger 460



22

22

et al., 2012). Several other transcriptional regulators are also required for this process, 461

such as chromatin-remodeling factors (Aichinger et al., 2012), histone 462

acetyltransferases, and splicing factors. In plants, Elongator might play a role in the 463

crosstalk between environmental and developmental stimuli to flexibly control SHR 464

transcription, thereby modulating root growth and development throughout the plants' 465

life cycle. 466

Materials and Methods 467

Plant Material and Growth Conditions 468

The Elongator mutants elp1 (abo1-2, SALK_004690), elp2, elp4 (SALK_079193), 469

elp6 and the double mutants elp1 elp2, elp1 elp4, elp1 elp6, elp2 elp4, elp2 elp6, and 470

elp4 elp6 (Zhou et al., 2009) were obtained from Zhizhong Gong. The mutants elp3 471

(elo3-6, GABI_555H06) (Nelissen et al., 2010) and elp5 (GABI_700A12) were 472

ordered from the Arabidopsis Biological Research Center. The plant materials used in 473

this study were previously described: plt1-4 plt2-2 (Aida et al., 2004), shr-2 474

(Helariutta et al., 2000), pCYCB1;1:GUS (Colon-Carmona et al., 1999), QC25 475

(Sabatini et al., 1999), pSHR:erGFP (Koizumi et al., 2012), pSHR:SHR-GFP 476

(Nakajima et al., 2001), pSCR:GFP-SCR (Sabatini et al., 1999), pCO2:H2B-YFP 477

(Heidstra et al., 2004), pCYCD6;1:GFP-GUS (Sozzani et al., 2010). The triple mutant 478

elp1 plt1-4 plt2-2, the double mutant elp1 shr-2, and different marker lines in the 479

mutant background were all obtained by genetic crossing. 480

Seeds of Arabidopsis, Arabidopsis thaliana (L.), were surface-sterilized with 10% 481

(v/v) bleach for 10 min and washed three times with sterile water. Sterilized seeds 482



23

23

were suspended in 0.1% (w/v) agarose and plated on half-strength Murashige and 483

Skoog (1/2 MS, PhytoTechnology Laboratories) medium. After stratification for 2 484

days at 4°C, they were transferred to the growth chamber at 22°C with a 16-h 485

light/8-h dark cycle. 486

487

Plasmid Construction and Plant Transformation 488

An approximately 2.0-kb fragment including the promoter region and the coding 489

sequences for the N-terminal 29 amino acids of ELP1 was amplified from genomic 490

DNA by PCR and cloned into the PacI/AscI sites of the binary vector pMDC162, 491

resulting in the pELP1:GUS construct, in which the coding sequences were fused in 492

frame with GUS. For the pELP3:ELP3-GFP construction, the region containing the 493

GFP-coding sequence and NOS-T fragment from the pGFP-2 vector, the CDS of 494

ELP3 and its promoter sequences were sequentially cloned in frame into the binary 495

vector pCAMBIA1300 with the restriction enzyme sites XbaI/BamHI and 496

SalI/XbaIrespectively.. For generation of the p35S:ELP3-myc or p35S:ELP3-GFP 497

plasmids, the ELP3 CDS was first cloned with the pENTR Directional TOPO Cloning 498

Kit (Invitrogen) and then recombined with the binary vector pGWB17 or pGWB5 with 499

the Gateway LR Clonase Enzyme Mix (Invitrogen). The p35S:NLS-ELP3-GFP 500

construct was generated same as p35S:ELP3-GFP except that the sequence encoding 501

the functional SV40 NLS (van der Krol and Chua, 1991) was attached to the forward 502

primer used for cloning the ELP3 CDS. Construction method for the 503

p35S:SPT4-2-GFP plasmid was the same as that for p35S:ELP3-GFP. For the 504



24

24

SPT4-RNAi construct, the SPT4-2 CDS was cloned into pHellsgate 2 in both forward 505

and reverse directions with a one-step BP reaction. All the primers used for the 506

molecular cloning are listed in Supplemental Table S1. 507

All the constructs were transformed into the Agrobacterium tumefaciens strain 508

GV3101 (pMP90), which was used for plant transformation with the vacuum 509

infiltration method. 510

Histology and Microscopy 511

Phenotypic analysis, Lugol staining, GUS staining, microscopic observation, and 512

confocal microscopy were all done as described previously (Zhou et al., 2010). For 513

marker expression control, at least 15 seedlings were used for each sample and 514

representative images were shown. For quantitative measurements, 20 seedlings of 515

each sample were analyzed and the statistical significance was evaluated by the 516

Student's t test. For multiple comparisons, an analysis of variance was followed by 517

Fisher's least significant difference test (SPSS) on the data. 518

519

Whole-Mount RNA in Situ Hybridization 520

Whole-mount RNA was hybridized in situ according to the method previously 521

described, and the probes for WOX5, PLT1, and PLT2 had already been synthesized 522

(Zhou et al., 2010). The antisense and sense probes for SHR were synthesized with 523

digoxigenin-11-UTP (Roche Diagnostics) by T7 RNA polymerase from an 524

SHR-specific fragment with the T7 promoter sequence either at the reverse primer or 525

at the forward primer, respectively (Supplemental Table S1). To enhance the probe 526



25

25

permeability for the SHR detection, SHR probes were hydrolyzed to an approximately 527

100-bp size, the proteinase K concentration was increased to 80 µg/mL, and the 528

incubation time was prolonged to 20 min. 529

530

Gene Expression Analysis 531

For RT-qPCR analysis, approximately 0.5-cm root tips were harvested from 5-day-old 532

seedlings for RNA extraction with TRIzol reagent (Invitrogen). First-strand cDNA 533

was synthesized from 2 µg of total RNA with the M-MLV reverse transcriptase 534

(Promega) and oligo(dT) primer and was quantified with the LightCycler 480 II 535

apparatus (Roche) and the SYBR Green Kit (Takara) according to the manufacturer's 536

instructions. The expression levels of the target genes were normalized to the 537

reference gene PP2AA3. The statistical significance was evaluated by Student's t test. 538

Primers used for RT-qPCR analysis are listed in Supplemental Table S1, of which 539

some had been described previously (Sozzani et al., 2010). 540

541

Cell Fractionation 542

Cell fractionation was performed with Plant Nuclei Isolation/Extraction Kit (Sigma). 543

Briefly, 4 g of 10-day-old WT seedlings was harvested and fully ground in liquid 544

nitrogen. The powder was transferred to 8 mL of precooled NIBA buffer (Sigma) and 545

filtered through nylon membrane. Triton X-100 was added to a final concentration of 546

0.5% (v/v) and the sample was kept on ice for 15 min, followed by centrifugation at 547

2,000×g at 4°C for 10 min. The extracts before centrifugation were collected as total 548



26

26

proteins (T), whereas the supernatants after centrifugation were collected as 549

cytoplasmic fractions (C). The pellets were resuspended in 1 mL of NIBA and applied 550

on top of a 800-μL cushion of 1.5 M sucrose, followed by centrifugation at 12,000×g 551

at 4°C for 10 min. The pellets were washed twice by resuspension in 1 mL of NIBA 552

and centrifugation at 12,000×g for 5 min. Thereafter, the pellets were resuspended in 553

600 µL of NIBA as the nuclear fraction (N). For each fraction, samples of 20 µL of 554

protein were used for immunoblot analysis. 555

556

Antibody Preparation 557

The partial CDS encoding the 400 amino acids of the C-terminus of ELP1 558

(BamHI/XhoI) and the full-length CDS of ELP3 (BamHI/HindIII) were cloned into 559

the pET-28a vector to express the recombinant proteins in Escherichia coli strain 560

BL21. The primers used for cloning are listed in Supplemental Table S1. The 561

recombinant proteins were used to raise polyclonal antibodies in mice. 562

563

Immunoblot Analysis 564

Protein extraction and immunoblot were done according to standard protocols. 565

Seedlings were ground into a fine powder in liquid nitrogen and then transferred to 566

extraction buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% [v/v] Nonidet P-40, 1 567

mM phenylmethylsulfonyl fluoride [PMSF], 10 µM MG132, and protease inhibitor 568

cocktail [Roche]). For immunoblot analysis, protein samples were boiled for 5 min 569

after mixing with sodium dodecyl sulfate (SDS) loading buffer, separated by 570



27

27

SDS-polyacrylamide gel electrophoresis, and transferred to polyvinylidene fluoride 571

membranes. Immunoblots were probed with the following antibodies: α-CTD (Abcam, 572

1:2000); α-H3 (Abcam, 1:4000); α-PEPC (Rockland, 1:2000); α-myc (Abmart, 573

1:2000); α-ELP1 (1:2000); and α-ELP3 (1:4000). Ponceau S-stained membranes were 574

shown as loading controls. 575

576

Y2H Assays 577

Y2H assays were based on the MATCHMAKER GAL4 Two-Hybrid System 578

(Clontech). The full-length CDS of each of the six Elongator subunits (SmaI/SacI) 579

was cloned into pGADT7, whereas the sequence encoding the RNAPII CTD 580

(EcoRI/BamHI) was cloned into pGBKT7. The primers used for cloning are listed in 581

Supplemental Table 1. Constructs were cotransformed into the yeast strain 582

Saccharomyces cerevisiae AH109. The presence of the transgenes was confirmed by 583

growth on SD/-Leu/-Trp plates. For protein interaction assessment, the transformed 584

yeast was suspended in liquid SD/-Leu/-Trp medium and cultured to an optical 585

density (OD) of 1.0. Five microliters of suspended yeast were dropped on plates 586

containing SD/-Ade/-His/-Leu/-Trp medium. Interactions were observed after 3 days 587

of incubation at 30°C. 588

589

LCI Assays 590

LCI assays were done with Nicotiana benthamiana leaves as previously described 591

(Song et al., 2011). Briefly, the full-length CDS of the two proteins were cloned into 592



28

28

pCAMBIA1300-NLUC (BamHI/SalI or SacI/SalI) and pCAMBIA1300-CLUC 593

(KpnI/SalI). Primers used for the vector construction are shown in Supplemental 594

Table S1. The resulting constructs were introduced into Agrobacterium strain 595

GV3101. The tobacco leaves were coinfiltrated with combinations of strains as 596

described and incubated for 3 days before observation with NightOWL II LB 983 597

(Berthold) imaging system. 598

599

Co-IP Assays 600

The Co-IP assays were done according to the published method (Chen et al., 2012) 601

with minor modifications. Briefly, total proteins were extracted from 10-day-old 602

Col-0 and p35S:SPT4-2-GFP seedlings with the protein lysis buffer (50 mM Tris-HCl, 603

pH 7.5, 150 mM NaCl, 0.1% [v/v] Triton X-100, 0.2% [v/v] Nonidet P-40, 0.6 mM 604

PMSF, 20 µM MG132, and protease inhibitor cocktail [Roche]). To preclear 2 mg of 605

protein extracts, 20 µL protein A/G plus agarose (Santa Cruz) was used. Thereafter, 606

the supernatants were incubated with 2 µL GFP antibody (Abcam) overnight and 607

further precipitated with another 20 µL protein A/G plus agarose (Santa Cruz). The 608

precipitated samples were washed four times with the lysis buffer and then eluted by 609

boiling for 5 min with SDS loading buffer. Immunoblots were detected with α-ELP1 610

(1:2000) and α-GFP (Abmart, 1:2000). 611

612

ChIP-qPCR Assays 613



29

29

ChIP assays were done according to the published protocol (Chen et al., 2012). 614

Briefly, 2 g of 5-day-old seedlings were cross-linked in 1% (v/v) formaldehyde for 615

chromatin isolation. For immunobinding, 2 µL CTD antibody (Abcam) or GFP 616

antibody (Abcam) was used. The protein-DNA complex was captured with 50 µL 617

protein A agarose/salmon sperm DNA (Millipore). The eluted DNA was purified with 618

QIAquick PCR purification kit (Qiagen) and used for qPCR analysis. Primers used for 619

ChIP-qPCR are listed in Supplemental Table S1. 620

621

RIP-PCR Assays 622

The RIP assays described previously (Zheng et al., 2009) were slightly modified. 623

Five-day-old seedlings of elp3;p35S:NLS-ELP3-GFP were harvested for RIP assays. 624

The seedlings were cross-linked in 1% (v/v) formaldehyde. Subsequently, 625

protein-RNA complexes were isolated and immunoprecipitated according to 626

published procedures. The associated RNAs were detected with semi-quantitative 627

reverse transcription PCR with primer pairs listed in Supplemental Table S1. 628

629

Accession Numbers 630

The sequence data can be found in the Arabidopsis Genome Initiative under the 631

following accession numbers: ELP1 (At5g13680), ELP2 (At1g49540), ELP3 632

(At5g50320), ELP4 (At3g11220), ELP5 (At2g18410), ELP6 (At4g10090), SHR 633

(AT4g37650), SCR (AT3g54220), BR6ox2 (AT3g30180), CYCD6;1 (At4g03270), 634

MGP (AT1g03840), NUC (AT5g44160), RLK (At5g67280), SNE ((At5g48170), 635



30

30

PP2AA3 (AT1g13320), PLT1 (At3g20840), PLT2 (At1g51190), WOX5 (At3g11260), 636

ACT7 (At5g09810), SPT4-1 (At5g08565), and SPT4-2 (At5g63670). 637

Supplemental Data 638

Supplemental Figure S1. Elongator acts as an integral complex to regulate root 639

growth. 640

Supplemental Figure S2. Elongator acts independently of the PLT pathway. 641

Supplemental Figure S3. Elongator functions in root radial patterning through the 642

SHR pathway. 643

Supplemental Figure S4. Subcellular localization of ELP3. 644

Supplemental Figure S5. Antibody characterization, cell fractionation assay and 645

histone acetylation detection. 646

Supplemental Figure S6. Construction of SPT4-RNAi. 647

Supplemental Table S1. List of primers used in this study. 648

649

Acknowledgments 650

We thank Zhizhong Gong, Ben Scheres, and Klaus Palme for sharing their research 651

materials and Martine De Cock for helping us prepare the manuscript. 652

653

654

Competing financial interests 655

The authors declare no competing financial interests. 656



31

31

Figure Legends 657

Figure 1. Elongator functions in root SCN maintenance and radial patterning through 658

the SHR pathway. A and B, Double staining of the QC-specific marker QC25 (blue) 659

and starch granules (dark brown) in the wild type (WT) (A) and elp1 (B) at 5 days 660

after germination (DAG). C and D, WOX5 expression in 5 DAG WT (C) and elp1 (D) 661

revealed by whole-mount RNA in situ hybridization with a WOX5 antisense probe. E, 662

Photographs of 5 DAG of WT, elp1, shr-2, and elp1 shr-2 seedlings showing the 663

involvement of the genetic relationship of Elongator and SHR in regulating root 664

growth. F, Quantification of meristem cell number of the indicated plants. Data shown 665

are average and SD (n = 20). Samples with different letters are significantly different 666

at P < 0.01 (Fisher’s LSD mean separation test). G to J, Expression of the 667

cortex-specific marker pCO2:H2B-YFP (G and H) and endodermis-specific marker 668

pSCR:GFP-SCR (I and J) in WT (G and I) and elp1 (H and J) roots. White rectangles 669

highlight the disorganized cell layers in the elp1 mutant, and horizontal white bars 670

indicate the stele width (including pericycle cells). K to N, Transverse confocal 671

sections showing the expression of the endodermis-specific marker pSCR:GFP-SCR 672

at the CEI/CEID position (K and M) and the transition zone (TZ) (L and N) in the WT 673

(K and L) and elp1 (M and N). Horizontal white bars indicate the stele width 674

(including pericycle cells). O, Quantification of stele width in WT and elp1. The stele 675

width (including the pericycle cells) at the TZ position in the longitudinal confocal 676

images was measured with ImageJ software. Data shown are average and SD (n = 20), 677



32

32

and asterisks denote Student’s t test significance compared with the WT: **P < 0.01. 678

Bars: A, B, and G to N, 50 µm; C and D, 20 µm. 679

Figure 2. The effect of Elongator depletion on the expression of SHR and its target 680

genes. A to F, SHR expression is compared between the elp1 mutant (B, D, and F) and 681

the WT (A, C, and E), as revealed by the expression of the marker constructs 682

pSHR:erGFP (A and B) and pSHR:SHR-GFP (C and D) and by whole-mount RNA in 683

situ hybridization with a SHR antisense probe (E and F). G, RT-qPCR analysis 684

showing the relative expression levels of SHR and its target genes in WT and elp1. 685

Total RNA was extracted from 0.5 cm root tip sections of 5 DAG seedlings. 686

Transcript levels were normalized to the reference gene PP2AA3. Error bars represent 687

SD (Student’s t test, **P < 0.01). The experiments were repeated three times, yielding 688

similar results. H and I, Representative images showing the location-specific 689

expression and reduced expression of pCYCD6;1:GFP-GUS in CEI/CEID cells of the 690

WT and the elp1 mutant, respectively. J, GUS staining of pELP1:GUS showing the 691

expression pattern of ELP1 in root tips. Data information: Bars in A to F, H, and I, 692

50 µm; J, 100 µm. 693

694

Figure 3. Importance of the nuclear localization of Elongator for its function. A and B, 695

Representative images showing the localization of ELP3-GFP in 5 DAG 696

elp3;pELP3:ELP3-GFP transgenic plants. (B) Magnification of the image in the 697

white rectangle in (A). C, Cell fractionation assay of Col-0 seedlings. Ten-day-old 698

Col-0 seedlings were collected for cell fractionation. Proteins from different fractions 699



33

33

were immunoblotted with antibodies against ELP1, ELP3, H3, and PEPC. H3 and 700

PEPC were used as nucleus- and cytoplasm-specific marker proteins, respectively. 701

Asterisk indicates the position of the specific band. T, total extracts; C, cytoplasmic 702

fraction; N, nuclear fraction. The experiments were repeated three times, yielding 703

similar results. D and E, Representative images showing the subcellular localization 704

of ELP3-GFP (D) and NLS-ELP3-GFP (E), indicating the nuclear localization of 705

NLS-ELP3-GFP. F, Photograph of 5 DAG seedlings showing that nuclear-localized 706

ELP3 fully complemented the short-root phenotype of the elp3 mutant. ELP3 was 707

fused with an efficient SV40 nuclear localization signal (NLS) to artificially 708

translocate it into the nucleus. The resulting constructs were transformed into the elp3 709

background to determine phenotype complementation. Data information: Bars in A, E, 710

and F, 50 µm; B, 10 µm. 711

712

Figure 4. Elongator functions as a transcription elongation factor to regulate SHR 713

expression. A, Interactions of different Elongator subunits with RNAPII CTD in a 714

yeast two-hybrid assay. The yeast transformants were dropped onto 715

SD/-Ade/-His/-Trp/-Leu (SD/-4) medium to assess protein–protein interactions. The 716

experiments were repeated three times with similar results. B and C, Firefly luciferase 717

complementation imaging assay showing the interaction of the ELP4 subunit with 718

RNAPII CTD in N. benthamiana. N. benthamiana leaves were infiltrated with 719

Agrobacterium containing the indicated construct pairs (B). The image was obtained 3 720

days after infiltration (C). The colored bar indicates the relative signal intensity. The 721



34

34

experiments were repeated three times, yielding similar results. D, Photographs of 5 722

DAG seedlings showing the insensitivity of the elp1 mutant to 6-AU. WT and elp1 723

seeds were sown on 1/2 MS medium without or with 0.5 mg/L 6-AU, and the plates 724

were photographed at 5 DAG. E, of RNAPII enrichments at various regions of the 725

SHR locus by ChIP-qPCR. Chromatin was extracted from Col-0 and elp1 seedlings at 726

5 DAG and precipitated with an anti-CTD antibody (Abcam). Precipitated DNA was 727

amplified with primers corresponding to the different regions of SHR as shown. The 728

“No Ab” (no antibody) precipitates served as negative controls. The ChIP signal was 729

quantified as the percentage of total input DNA by qPCR and arbitrarily set to 1 in the 730

“No Ab” samples. TSS indicates transcription start site. The experiments were 731

repeated three times, yielding similar results. Error bars represent SD. Asterisks 732

indicate significant differences between Col-0 and the elp1 mutant, according to 733

Student’s t test (**P < 0.01). F, RIP-PCR results showing the association of Elongator 734

with SHR mRNA, but not with PLT2 or SCR mRNA. Protein-RNA complexes were 735

isolated from Col-0 and elp3;p35S:NLS-ELP3-GFP seedlings at 5 DAG and 736

precipitated with an anti-GFP antibody (Abcam). The precipitated RNA was reverse 737

transcribed and then amplified with primers targeting the respective CDS regions. The 738

RIP signal was quantified as the percentage of total input RNA by qPCR. Samples 739

before precipitation were taken as “Input”, and the “NA” (no antibody) precipitates 740

served as negative controls. The experiments were repeated three times with similar 741

results. 742

743



35

35

Figure 5. Elongator functions in concert with SPT4/SPT5. A, Co-IP assay showing 744

that Elongator associates with SPT4-2 in plant cells. Protein extracts from 10-day-old 745

Col-0 and p35S:SPT4-2-GFP seedlings were immunoprecipitated with an anti-GFP 746

antibody (Abcam). Samples before (Input) and after immunoprecipitation (IP) were 747

blotted with anti-GFP and anti-ELP1 antibodies. The experiments were repeated three 748

times with similar results. B and C, Firefly luciferase complementation imaging assay 749

showing the interaction of the ELP1 subunit with SPT4-2 in N. benthamiana. N. 750

benthamiana leaves were infiltrated with Agrobacterium strains containing the 751

indicated construct pairs (B). The image was obtained 3 days after infiltration (C). 752

The colored bar indicates the relative signal intensity. The experiments were repeated 753

three times, yielding similar results. D, Photographs of 5 DAG seedlings showing the 754

insensitivity of SPT4-RNAi to 6-AU. WT and SPT4-RNAi seeds were sown on 1/2 MS 755

medium without or with 0.5 mg/L 6-AU, and the plates were photographed at 5 DAG. 756

E, ChIP-qPCR results showing the enrichment of SPT4-2 on the SHR locus. Sonicated 757

chromatin from 5 DAG Col-0 and p35S:SPT4-2-GFP seedlings was precipitated with 758

an anti-GFP antibody (Abcam). The precipitated DNA was used as template for qPCR 759

analysis with primers targeting different regions of the SHR locus as shown. The 760

promoter region of ACT7 (ACT7-P) was used as a negative control. The ChIP signal 761

was quantified as the percentage of the total input DNA and was arbitrarily set to 1 in 762

Col-0. TSS, transcription start site. The experiments were repeated three times, 763

yielding similar results. Error bars represent SD. Asterisks indicate significant 764

differences, according to Student’s t test, **P < 0.01. F, Proposed mechanism in 765



36

36

which Elongator acts in concert with SPT4/SPT5 to regulate the transcription 766

elongation of SHR. During the transcription elongation process of SHR, Elongator 767

directly interacts with the RNAPII CTD and the nascent SHR mRNA and associates 768

with SPT4/SPT5. Meanwhile, SPT4/SPT5 is in close contact with the chromosomal 769

region harboring the SHR locus, the RNAPII subunits, and Elongator. 770

771

772

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Aichinger E, Kornet N, Friedrich T, Laux T (2012) Plant Stem Cell Niches. Annu Rev 775

Plant Biol 63: 615-636 776

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Figure 1.

WT QC25 elp1 QC25 WT elp1 WOX5 WOX5

C D A B

0

10

20

30

40

50

60

Ste

le w

idth

(μ

m)

WT WT

elp1 elp1

pSCR:GFP-SCR pSCR:GFP-SCR

pSCR:GFP-SCR pSCR:GFP-SCR

K L

M N

WT pSCR:GFP-SCR

I

elp1 pSCR:GFP-SCR

J

pCO2:H2B-YFP WT

G

pCO2:H2B-YFP elp1

H

O

WT elp1

**

WT elp1 shr-2 elp1 shr-2

E

0

5

10

15

20

25

30

35

40

45

Meri

ste

m c

ell

nu

mb

er

WT elp1 shr-2 elp1 shr-2

a

b

c c

F



Figure 1. Elongator functions in root SCN maintenance and radial patterning through the SHR pathway. A and B,

Double staining of the QC-specific marker QC25 (blue) and starch granules (dark brown) in the wild type (WT) (A)

and elp1 (B)at 5 days after germination (DAG). C and D, WOX5 expression in 5 DAG WT (C) and elp1 (D) revealed

by whole-mount RNA in situ hybridization with a WOX5 antisense probe. E, Photographs of 5 DAG of WT, elp1,

shr-2, and elp1 shr-2 seedlings showing the involvement of the genetic relationship of Elongator and SHR in

regulating root growth. F, Quantification of meristem cell number of the indicated plants. Data shown are average

and SD (n = 20). Samples with different letters are significantly different at P < 0.01(Fisher’s LSD mean separation

test). G to J, Expression of the cortex-specific marker pCO2:H2B-YFP (G and H) and endodermis-specific marker

pSCR:GFP-SCR (I and J) in WT (G and I) and elp1(H and J) roots. White rectangles highlight the disorganized cell

layers in the elp1 mutant, and horizontal white bars indicate the stele width (including pericycle cells). K to N,

Transverse confocal sections showing the expression of the endodermis-specific marker pSCR:GFP-SCR at the

CEI/CEID position (K and M) and the transition zone (TZ) (L and N) in the WT (K and L) and elp1 (M and N).

Horizontal white bars indicate the stele width (including pericycle cells). O, Quantification of stele width in WT and

elp1. The stele width (including the pericycle cells) at the TZ position in the longitudinal confocal images was

measured with ImageJ software. Data shown are average and SD (n = 20), and asterisks denote Student’s t test

significance compared with the WT: **P < 0.01. Bars: A, B, and G to N, 50 µm; C and D, 20 µm.



Figure 2.

W

T abo

1

WT

pSHR:erGFP

A

WT pSHR:SHR-GFP

C

WT SHR

E

elp1 pSHR:erGFP

B

elp1 pSHR:SHR-GFP

D

elp1 SHR

F

pELP1:GUS

J

WT pCYCD6;1:GFP-GUS

H

elp1 pCYCD6;1:GFP-GUS

I

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

SHR SCR Br6OX CYCD6;1 MGP NUC RLK SNE

Rela

tive e

xp

ressio

n

Col-0elp1

**

**

**

**

**

**

**

**

G

Figure 2. The effect of Elongator depletion on the expression of SHR and its target genes. A to F, SHR expression

is compared between the elp1 mutant (B, D, and F) and the WT (A, C, and E), as revealed by the expression of

the marker constructs pSHR:erGFP (A and B) and pSHR:SHR-GFP (C and D) and by whole-mount RNA in situ

hybridization with a SHR antisense probe (E and F). G, RT-qPCR analysis showing the relative expression levels of

SHR and its target genes in WT and elp1. Total RNA was extracted from 0.5 cm root tip sections of 5 DAG

seedlings. Transcript levels were normalized to the reference gene PP2AA3. Error bars represent SD (Student’s t

test, **P < 0.01). The experiments were repeated three times, yielding similar results. H and I, Representative

images showing the location-specific expression and reduced expression of pCYCD6;1:GFP-GUS in CEI/CEID

cells of the WT and the elp1 mutant, respectively. J, GUS staining of pELP1:GUS showing the expression pattern

of ELP1 in root tips. Data information: Bars in A to F, H, and I, 50 µm; J, 100 µm.



Figure 3.

elp3;pELP3:ELP3-GFP

A B

PEPC

ELP1

ELP3

*

T C N

H3

*

C

F

ELP3-GFP NLS-ELP3-GFP

D E

Figure 3. Importance of the nuclear localization of Elongator for its function. A and B, Representative images

showing the localization of ELP3-GFP in 5 DAG elp3;pELP3:ELP3-GFP transgenic plants. (B) Magnification of the

image in the white rectangle in (A). C, Cell fractionation assay of Col-0 seedlings. Ten-day-old Col-0 seedlings were

collected for cell fractionation. Proteins from different fractions were immunoblotted with antibodies against ELP1,

ELP3, H3, and PEPC. H3 and PEPC were used as nucleus- and cytoplasm-specific marker proteins, respectively.

Asterisk indicates the position of the specific band. T, total extracts; C, cytoplasmic fraction; N, nuclear fraction. The

experiments were repeated three times, yielding similar results. D and E, Representative images showing the

subcellular localization of ELP3-GFP (D) and NLS-ELP3-GFP (E), indicating the nuclear localization of NLS-ELP3-

GFP. F, Photograph of 5 DAG seedlings showing that nuclear-localized ELP3 fully complemented the short-root

phenotype of the elp3 mutant. ELP3 was fused with an efficient SV40 nuclear localization signal (NLS) to artificially

translocate it into the nucleus. The resulting constructs were transformed into the elp3 background to determine

phenotype complementation. Data information: Bars in A, E, and F, 50 µm; B, 10 µm.



Figure 4.

ELP1-AD+CTD-BD

ELP2-AD+CTD-BD

ELP3-AD+CTD-BD

ELP4-AD+CTD-BD

ELP5-AD+CTD-BD

ELP6-AD+CTD-BD

AD+CTD-BD

ELP1-AD+BD

ELP2-AD+BD

ELP3-AD+BD

ELP4-AD+BD

ELP5-AD+BD

ELP6-AD+BD

AD+BD

SD/-4

nLUC+

cLUC

nLUC+

cLuc-CTD

ELP4-nLUC

+cLUC

ELP4-nLUC+

cLUC-CTD

A B

Control

6-AU (5 μΜ)

WT elp1

B C

D E

F

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

1.80

ACT7 PLT2 SCR SHR

Rela

tive e

nri

ch

men

t

Col-0 NA

Col-0 GFP

NLS-ELP3-GFP NA

NLS-ELP3-GFP GFP

***

A B C P

SHR

5' 3'

TSS

0

2

4

6

8

10

12

14

16

SHR-P SHR-A SHR-B SHR-C

Rela

tive e

nri

ch

men

t

**

Col-0 No Ab

Col-0 Anti-CTD

elp1 Anti-CTD

elp1 No Ab

**

**

**

-201 -61 340 529 811 945 1236 1391



Figure 4. Elongator functions as a transcription elongation factor to regulate SHR expression. A, Interactions of

different Elongator subunits with RNAPII CTD in a yeast two-hybrid assay. The yeast transformants were dropped

onto SD/-Ade/-His/-Trp/-Leu (SD/-4) medium to assess protein–protein interactions. The experiments were

repeated three times with similar results. B and C, Firefly luciferase complementation imaging assay showing the

interaction of the ELP4 subunit with RNAPII CTD in N. benthamiana. N. benthamiana leaves were infiltrated with

Agrobacterium containing the indicated construct pairs (B). The image was obtained 3 days after infiltration (C).

The colored bar indicates the relative signal intensity. The experiments were repeated three times, yielding similar

results. D, Photographs of 5 DAG seedlings showing the insensitivity of the elp1 mutant to 6-AU. WT and elp1

seeds were sown on 1/2 MS medium without or with 0.5 mg/L 6-AU, and the plates were photographed at 5 DAG.

E, RNAPII enrichments at various regions of the SHR locus by ChIP-qPCR. Chromatin was extracted from Col-0

and elp1 seedlings at 5 DAG and precipitated with an anti-CTD antibody (Abcam). Precipitated DNA was amplified

with primers corresponding to the different regions of SHR as shown. The “No Ab” (no antibody) precipitates served

as negative controls. The ChIP signal was quantified as the percentage of total input DNA by qPCR and arbitrarily

set to 1 in the “No Ab” samples. TSS indicates transcription start site. The experiments were repeated three times,

yielding similar results. Error bars represent SD. Asterisks indicate significant differences between Col-0 and the

elp1 mutant, according to Student’s t test (**P < 0.01). F, RIP-PCR results showing the association of Elongator

with SHR mRNA, but not with PLT2 or SCR mRNA. Protein-RNA complexes were isolated from Col-0 and

elp3;p35S:NLS-ELP3-GFP seedlings at 5 DAG and precipitated with an anti-GFP antibody (Abcam). The

precipitated RNA was reverse transcribed and then amplified with primers targeting the respective CDS regions.

The RIP signal was quantified as the percentage of total input RNA by qPCR. Samples before precipitation were

taken as “Input”, and the “NA” (no antibody) precipitates served as negative controls. The experiments were

repeated three times with similar results.



Figure 5

ELP1 IP

ELP1 Input

GFP IP

nLUC+

cLUC nLUC+

cLUC-SPT4-2

ELP1-nLUC

+cLUC

ELP1-nLUC+

cLUC-SPT4-2

GFP Input

Control

0.5 mg/L 6-AU

WT SPT4-RNAi

A B C

D

E

F

0

2

4

6

8

10

ACT7-P SHR-P SHR-A SHR-B SHR-C

Rela

tive e

nri

ch

men

t Col-0

p35S:SPT4-2-GFP

**

**

** **

A B C

SHR

5' 3'

P

TSS

-201 -61 340 529 811 945 1236 1391



Figure 5. Elongator functions in concert with SPT4/SPT5. A, Co-IP assay showing that Elongator associates with

SPT4-2 in plant cells. Protein extracts from 10-day-old Col-0 and p35S:SPT4-2-GFP seedlings were

immunoprecipitated with an anti-GFP antibody (Abcam). Samples before (Input) and after immunoprecipitation (IP)

were blotted with anti-GFP and anti-ELP1 antibodies. The experiments were repeated three times with similar results.

B and C, Firefly luciferase complementation imaging assay showing the interaction of the ELP1 subunit with SPT4-2

in N. benthamiana. N. benthamiana leaves were infiltrated with Agrobacterium strains containing the indicated

construct pairs (B). The image was obtained 3 days after infiltration (C). The colored bar indicates the relative signal

intensity. The experiments were repeated three times, yielding similar results. D, Photographs of 5 DAG seedlings

showing the insensitivity of SPT4-RNAi to 6-AU. WT and SPT4-RNAi seeds were sown on 1/2 MS medium without

or with 0.5 mg/L 6-AU, and the plates were photographed at 5 DAG. E, ChIP-qPCR results showing the enrichment

of SPT4-2 on the SHR locus. Sonicated chromatin from 5 DAG Col-0 and p35S:SPT4-2-GFP seedlings was

precipitated with an anti-GFP antibody (Abcam). The precipitated DNA was used as template for qPCR analysis with

primers targeting different regions of the SHR locus as shown. The promoter region of ACT7 (ACT7-P) was used as

a negative control. The ChIP signal was quantified as the percentage of the total input DNA and was arbitrarily set to

1 in Col-0. TSS, transcription start site. The experiments were repeated three times, yielding similar results. Error

bars represent SD. Asterisks indicate significant differences, according to Student’s t test, **P < 0.01. F, Proposed

mechanism in which Elongator acts in concert with SPT4/SPT5 to regulate the transcription elongation of SHR.

During the transcription elongation process of SHR, Elongator directly interacts with the RNAPII CTD and the

nascent SHR mRNA and associates with SPT4/SPT5. Meanwhile, SPT4/SPT5 is in close contact with the

chromosomal region harboring the SHR locus, the RNAPII subunits, and Elongator.



Parsed CitationsAichinger E, Kornet N, Friedrich T, Laux T (2012) Plant Stem Cell Niches. Annu Rev Plant Biol 63: 615-636

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Aida M, Beis D, Heidstra R, Willemsen V, Blilou I, Galinha C, Nussaume L, Noh Y-S, Amasino R, Scheres B (2004) The PLETHORAgenes mediate patterning of the Arabidopsis root stem cell niche. Cell 119: 109-120


Anderson SL, Coli R, Daly IW, Kichula EA, Rork MJ, Volpi SA, Ekstein J, Rubin BY (2001) Familial dysautonomia is caused by mutationsof the IKAP gene. Am J Hum Genet 68: 753-758


Antosz W, Pfab A, Ehrnsberger HF, Holzinger P, Köllen K, Mortensen SA, Bruckmann A, Schubert T, Längst G, Griesenbeck J, et al(2017) The composition of the Arabidopsis RNA Polymerase II transcript elongation complex reveals the interplay between elongationand mRNA processing factors. Plant Cell 29: 854-870


Bauer F, Matsuyama A, Candiracci J, Dieu M, Scheliga J, Wolf Dieter A, Yoshida M, Hermand D (2012) Translational control of celldivision by Elongator. Cell Rep 1: 424-433


Chen C, Huang B, Eliasson M, Rydén P, Byström AS (2011) Elongator complex influences telomeric gene silencing and DNA damageresponse by its role in wobble uridine tRNA modification. PLoS Genet 7: e1002258


Chen R, Jiang H, Li L, Zhai Q, Qi L, Zhou W, Liu X, Li H, Zheng W, Sun J, et al (2012) The Arabidopsis Mediator subunit MED25differentially regulates jasmonate and abscisic acid signaling through interacting with the MYC2 and ABI5 transcription factors. PlantCell 24: 2898-2916


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Colon-Carmona A, You R, Haimovitch-Gal T, Doerner P (1999) Technical advance: spatio-temporal analysis of mitotic activity with alabile cyclin-GUS fusion protein. Plant J 20: 503-508


Creppe C, Malinouskaya L, Volvert M-L, Gillard M, Close P, Malaise O, Laguesse S, Cornez I, Rahmouni S, Ormenese S, et al (2009)Elongator controls the migration and differentiation of cortical neurons through acetylation of α-Tubulin. Cell 136: 551-564


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Di Laurenzio L, Wysocka-Diller J, Malamy JE, Pysh L, Helariutta Y, Freshour G, Hahn MG, Feldmann KA, Benfey PN (1996) TheSCARECROW gene regulates an asymmetric cell division that is essential for generating the radial organization of the Arabidopsisroot. Cell 86: 423-433


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https://www.ncbi.nlm.nih.gov/pubmed?term=Aichinger E%2C Kornet N%2C Friedrich T%2C Laux T %282012%29 Plant Stem Cell Niches%2E Annu Rev Plant Biol&dopt=abstract

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https://www.ncbi.nlm.nih.gov/pubmed?term=Chen R%2C Jiang H%2C Li L%2C Zhai Q%2C Qi L%2C Zhou W%2C Liu X%2C Li H%2C Zheng W%2C Sun J%2C et al %282012%29 The Arabidopsis Mediator subunit MED25 differentially regulates jasmonate and abscisic acid signaling through interacting with the MYC2 and ABI5 transcription factors%2E Plant Cell&dopt=abstract


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https://www.ncbi.nlm.nih.gov/pubmed?term=Esberg A%2C Huang B%2C Johansson MJ%2C Bystr�m AS %282006%29 Elevated levels of two tRNA Species bypass the requirement for Elongator complex in transcription and exocytosis%2E Mol Cell&dopt=abstract

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https://www.ncbi.nlm.nih.gov/pubmed?term=Exinger F%2C Lacroute F %281992%29 6%2DAzauracil inhibition of GTP biosynthesis in Saccharomyces cerevisiae%2E Curr Genet&dopt=abstract

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https://www.ncbi.nlm.nih.gov/pubmed?term=Fern�ndez%2DV�zquez J%2C Vargas%2DP�rez I%2C Sans� M%2C Buhne K%2C Carmona M%2C Paulo E%2C Hermand D%2C Rodr�guez%2DGabriel M%2C Ayt� J%2C Leidel S%2C et al %282013%29 Modification of tRNALysUUU by Elongator is essential for efficient translation of stress mRNAs%2E PLoS Genet 9%3A e&dopt=abstract

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https://scholar.google.com/scholar?as_q=Elongator complex: how many roles does it play&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Svejstrup&as_ylo=2007&as_allsubj=all&hl=en&c2coff=1

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https://scholar.google.com/scholar?as_q=Cell fate in the Arabidopsis root meristem determined by directional signalling&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=van&as_ylo=1995&as_allsubj=all&hl=en&c2coff=1

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https://www.ncbi.nlm.nih.gov/pubmed?term=Wang Y%2C An C%2C Zhang X%2C Yao J%2C Zhang Y%2C Sun Y%2C Yu F%2C Amador DM%2C Mou Z %282013%29 The Arabidopsis Elongator complex Subunit2 Epigenetically Regulates Plant Immune Responses%2E Plant Cell&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Wang&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=The Arabidopsis Elongator complex Subunit2 Epigenetically Regulates Plant Immune Responses&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=The Arabidopsis Elongator complex Subunit2 Epigenetically Regulates Plant Immune Responses&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Wang&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Welch D%2C Hassan H%2C Blilou I%2C Immink R%2C Heidstra R%2C Scheres B %282007%29 Arabidopsis JACKDAW and MAGPIE zinc finger proteins delimit asymmetric cell division and stabilize tissue boundaries by restricting SHORT%2DROOT action%2E Genes Dev&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Welch&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Arabidopsis JACKDAW and MAGPIE zinc finger proteins delimit asymmetric cell division and stabilize tissue boundaries by restricting SHORT-ROOT action&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Arabidopsis JACKDAW and MAGPIE zinc finger proteins delimit asymmetric cell division and stabilize tissue boundaries by restricting SHORT-ROOT action&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Welch&as_ylo=2007&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Winkler GS%2C Kristjuhan A%2C Erdjument%2DBromage H%2C Tempst P%2C Svejstrup JQ %282002%29 Elongator is a histone H3 and H4 acetyltransferase important for normal histone acetylation levels in vivo%2E Proc Natl Acad Sci USA&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Winkler&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Elongator is a histone H3 and H4 acetyltransferase important for normal histone acetylation levels in vivo&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Elongator is a histone H3 and H4 acetyltransferase important for normal histone acetylation levels in vivo&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Winkler&as_ylo=2002&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Wittschieben BO%2C Otero G%2C de Bizemont T%2C Fellows J%2C Erdjument%2DBromage H%2C Ohba R%2C Li Y%2C Allis CD%2C Tempst P%2C Svejstrup JQ %281999%29 A novel histone acetyltransferase is an integral subunit of elongating RNA polymerase II holoenzyme%2E Mol Cell&dopt=abstract

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https://scholar.google.com/scholar?as_q=A novel histone acetyltransferase is an integral subunit of elongating RNA polymerase II holoenzyme&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=A novel histone acetyltransferase is an integral subunit of elongating RNA polymerase II holoenzyme&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Wittschieben&as_ylo=1999&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Woloszynska M%2C Le Gall S%2C Van Lijsebettens M %282016%29 Plant Elongator%2Dmediated transcriptional control in a chromatin and epigenetic context%2E Biochim Biophys Acta&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Woloszynska&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Plant Elongator-mediated transcriptional control in a chromatin and epigenetic context&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Plant Elongator-mediated transcriptional control in a chromatin and epigenetic context&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Woloszynska&as_ylo=2016&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Wu X%2C Rossettini A%2C Hanes SD %282003%29 The ESS1 prolyl isomerase and its suppressor BYE1 interact with RNA pol II to inhibit transcription elongation in Saccharomyces cerevisiae%2E Genetics&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Wu&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=The ESS1 prolyl isomerase and its suppressor BYE1 interact with RNA pol II to inhibit transcription elongation in Saccharomyces cerevisiae&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=The ESS1 prolyl isomerase and its suppressor BYE1 interact with RNA pol II to inhibit transcription elongation in Saccharomyces cerevisiae&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Wu&as_ylo=2003&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Yamaguchi Y%2C Shibata H%2C Handa H %282013%29 Transcription elongation factors DSIF and NELF%3A promoter%2Dproximal pausing and beyond%2E Biochim Biophys Acta&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Yamaguchi&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Transcription elongation factors DSIF and NELF: promoter-proximal pausing and beyond&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Transcription elongation factors DSIF and NELF: promoter-proximal pausing and beyond&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Yamaguchi&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Zhang X%2C Zhou W%2C Chen Q%2C Fang M%2C Zheng S%2C Scheres B%2C Li C %282018%29%2E Mediator subunit MED31 is required for radial patterning of Arabidopsis roots%2E Proc Natl Acad Sci USA 115%3A E5624%2DE&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Zhang&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Mediator subunit MED31 is required for radial patterning of Arabidopsis roots&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Mediator subunit MED31 is required for radial patterning of Arabidopsis roots&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Zhang&as_ylo=2018&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Zheng B%2C Wang Z%2C Li S%2C Yu B%2C Liu JY%2C Chen X %282009%29 Intergenic transcription by RNA polymerase II coordinates Pol IV and Pol V in siRNA%2Ddirected transcriptional gene silencing in Arabidopsis%2E Genes Dev&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Zheng&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Intergenic transcription by RNA polymerase II coordinates Pol IV and Pol V in siRNA-directed transcriptional gene silencing in Arabidopsis&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Intergenic transcription by RNA polymerase II coordinates Pol IV and Pol V in siRNA-directed transcriptional gene silencing in Arabidopsis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Zheng&as_ylo=2009&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Zhou W%2C Wei L%2C Xu J%2C Zhai Q%2C Jiang H%2C Chen R%2C Chen Q%2C Sun J%2C Chu J%2C Zhu L%2C et al %282010%29 Arabidopsis Tyrosylprotein sulfotransferase acts in the auxin%2FPLETHORA pathway in regulating postembryonic maintenance of the root stem cell niche%2E Plant Cell&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Zhou&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Arabidopsis Tyrosylprotein sulfotransferase acts in the auxin/PLETHORA pathway in regulating postembryonic maintenance of the root stem cell niche&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Arabidopsis Tyrosylprotein sulfotransferase acts in the auxin/PLETHORA pathway in regulating postembryonic maintenance of the root stem cell niche&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Zhou&as_ylo=2010&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Zhou X%2C Hua D%2C Chen Z%2C Zhou Z%2C Gong Z %282009%29 Elongator mediates ABA responses%2C oxidative stress resistance and anthocyanin biosynthesis in Arabidopsis%2E Plant J&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Zhou&hl=en&c2coff=1

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https://scholar.google.com/scholar?as_q=Elongator mediates ABA responses, oxidative stress resistance and anthocyanin biosynthesis in Arabidopsis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Zhou&as_ylo=2009&as_allsubj=all&hl=en&c2coff=1


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Elongator is required for root stem cell maintenance by ... · 11/6/2018 · 102 and ELP2 functioning as scaffolds for complex assembly, ELP3 acting as the catalytic 103 subunit,