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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
11
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
17
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
28
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
48
Abstract 49
50
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
66
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Introduction 67
68
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
135
Results 136
137
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
193
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
217
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
255
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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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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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
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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.
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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.
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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.
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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
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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.
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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
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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.
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