1 running head: tetraspanin genes in arabidopsis … · 103 in primary root growth and lateral root...
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Running head: TETRASPANIN genes in Arabidopsis development 1
Author for correspondence: 2
Mieke Van Lijsebettens 3
Department of Plant Systems Biology 4
VIB-Ghent University 5
Technologiepark 927 6
B-9052 Gent (Belgium) 7
Tel.: +32 9 3313970; Fax: +32 9 3313809 8
E-mail: [email protected] 9
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Journal research area: Genes, Development and Evolution: Associate Editors Jiming Jiang 11
(Madison) and Hong Ma (Shanghai and University Park) 12
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Plant Physiology Preview. Published on September 28, 2015, as DOI:10.1104/pp.15.01310
Copyright 2015 by the American Society of Plant Biologists
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Functional Analysis of Arabidopsis TETRASPANIN Gene Family in Plant Growth and 14
Development1[OPEN] 15
Feng Wang, Antonella Muto, Jan Van de Velde, Pia Neyt, Kristiina Himanen2, Klaas 16
Vandepoele, and Mieke Van Lijsebettens* 17
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Department of Plant Systems Biology, VIB, B-9052 Ghent, Belgium (F.W, A.M., J.V.d.V., P.N., 19
K.H., K.V., M.V.L.); Department of Plant Biotechnology and Bioinformatics, Ghent University, 20
B-9052 Ghent, Belgium (F.W, A.M., J.V.d.V., P.N., K.H., K.V., M.V.L.); and Department of 21
Biology, Ecology and Earth Sciences, University of Calabria, 87036 Arcavacata of Rende, Italy 22
(A.M.) 23
ORCID KV 0000-0003-4790-2725, JVDV 0000-0001-7742-1266, MVL 0000-0002-7632-1463. 24
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One-sentence summary: TETRASPANIN gene expression patterns and mutational analysis 26
inferred function in plant growth and development, which was furthered by cis regulatory 27
elements and a TF-TET regulatory network 28
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1 This work was supported by the China Scholarship Council (CSC) (predoctoral fellowship to 30
F.W.), and by the Ministero dell' Istruzione, dell'Università e della Ricerca (MIUR) 31
(predoctoral fellowship to A.M.). KV acknowledges the Multidisciplinary Research 32
Partnership “Bioinformatics: from nucleotides to networks” Project (no 01MR0310W) of 33
Ghent University. J.VdV. is indebted to the Agency for Innovation by Science and Technology 34
(IWT) in Flanders for a predoctoral fellowship. 35 2 Present address: Department of Agricultural Sciences, University of Helsinki, 000014 Helsinki, 36
Finland 37 * Address correspondence to [email protected]. 38
The author responsible for distribution of materials integral to the findings presented in this 39
article in accordance with the policy described in the Instructions for Authors 40
(www.plantphysiol.org) is: Mieke Van Lijsebettens ([email protected]). 41 [OPEN] Articles can be viewed without a subscription. 42
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Abstract 44
TETRASPANIN (TET) genes encode conserved integral membrane proteins that are known in 45
animals to function in cellular communication during gamete fusion, immunity reaction and 46
pathogen recognition. In plants, functional information is limited to one of the 17 members of the 47
Arabidopsis TET gene family and to expression data in reproductive stages. Here, the promoter 48
activity of all 17 Arabidopsis TET genes was investigated by pAtTET::NLS-GFP/GUS reporter 49
lines throughout the life cycle, which predicted functional divergence in the paralogous genes per 50
clade. However, partial overlap was observed for many TET genes across the clades, correlating 51
with few phenotypes in single mutants and therefore requiring double mutant combinations for 52
functional investigation. Mutational analysis showed a role for TET13 in primary root growth and 53
lateral root development, and redundant roles for TET5 and TET6 in leaf and root growth through 54
negative regulation of cell proliferation. Strikingly, a number of TET genes were expressed in 55
embryonic and seedling progenitor cells and remained expressed until the differentiation state in 56
the mature plant, suggesting a dynamic function over developmental stages. cis-regulatory 57
elements together with transcription factor binding data provided molecular insight into the site, 58
conditions and perturbations that affect TET gene expression, and positioned the TET genes in 59
different molecular pathways; the data represent a hypothesis-generating resource for further 60
functional analyses. 61
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Key words: Arabidopsis; tetraspanin; gene duplication; lateral root development; vascular tissue; 63
promoter elements; transcription factor regulatory network 64
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During embryogenesis in plants, the fertilized egg cell develops gradually by consecutive, but 67
partially overlapping, processes, such as cell division, patterning and growth, into the 68
rudimentary body plan of the mature embryo. Early pattern formation in the embryo generates the 69
body plan polarity with apical and basal stem cell progenitor domains and the different concentric 70
progenitor tissue layers, in which cells interpret their position, acquire cell fate and differentiate 71
into specific morphologies and functions after germination. Then, the apical meristems are 72
activated to generate the primary root and the above-ground vegetative structures (Murray et al., 73
2012; Perilli et al., 2012). In the seedling, patterning processes occur in the epidermis to generate 74
specialized cells such as trichomes and stomata, in the shoot apical meristem (SAM) upon leaf 75
initiation and in the primary root pericycle upon lateral root initiation. Developmental programs 76
such as germination, photomorphogenesis and floral transition occur in response to 77
environmental stimuli, such as light, circadian clock and temperature, and hormonal stimuli that 78
require cellular communication and signaling. Membrane proteins, located at the plasma 79
membrane, are the most upstream components in signaling perception and transduction during 80
cellular communication. It is estimated that 20 to 30% of all genes in most genomes encode 81
integral proteins (Krogh et al., 2001), which are transmembrane proteins. 82
Tetraspanins are a distinct class of conserved integral proteins with four transmembrane 83
domains, a small extracellular loop and a large, cystein-rich extracellular loop, which is important 84
for interacting with each other and other proteins to form tetraspanin-enriched microdomains that 85
participate in cell-to-cell communication processes during cell morphogenesis, motility and 86
fusion (Yáñez-Mó et al., 2009). Tetraspanins comprise large gene families in multicellular 87
organisms; 33 in human, and 36 in Drosophila melanogaster (Huang et al., 2005). Mutations in 88
animal tetraspanins result in severe defects, such as incurable blindness and impaired fertility 89
(Kaji et al., 2002; Goldberg, 2006). In plants, phylogenetic studies identified 17 tetraspanin genes 90
in the Arabidopsis genome, most of which are duplicated (Cnops et al., 2006; Wang et al., 2012; 91
Boavida et al., 2013). Mutants of the Arabidopsis TETRASPANIN1/TORNADO2/EKEKO 92
(TET1/TRN2) show defects in leaf lamina symmetry and venation pattern, root epidermal 93
patterning (Cnops et al., 2000; Cnops et al., 2006), peripheral zone identity of the SAM (Chiu et 94
al., 2007) and megasporogenesis (Lieber et al., 2011). A number of Arabidopsis tetraspanins are 95
expressed in reproductive tissues and at fertilization, and are localized at the plasma membrane of 96
protoplasts (Boavida et al., 2013). However, a systematic analysis of all members of the plant 97
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TET gene family in embryonic and vegetative development and upon hormonal and 98
environmental stimuli is lacking. 99
Here, the 17 members of the Arabidopsis TET gene family were analyzed for their cellular 100
expression during embryonic and vegetative development by pAtTET1-17::NLS-GFP/GUS 101
reporter lines, which was instrumental for further mutational analyses. Indeed, a role for TET13 102
in primary root growth and lateral root development, and redundant roles for TET5 and TET6 in 103
leaf and root growth were identified, as was suggested by the site of their expression. 104
Complementary experimental and computational regulatory datasets were integrated to identify 105
putative cis-regulatory elements in the TET promoters. A transcription factor (TF)-TET gene 106
regulatory network was delineated and regulatory interactions were confirmed by perturbation 107
expression data. 108
RESULTS 109
Expression of TET Genes in Specific Tissues, Domains and Cells throughout the Plant’s Life 110
Cycle 111
The promoter activities of the 17 TET gene family members were analyzed by pAtTET::NLS-112
GFP/GUS lines throughout the life cycle in order to reveal their site and timing of activity and 113
help in designing further mutational analyses. Per TET gene, at least three independent T2 114
pAtTET::NLS-GFP/GUS lines, with a single-locus insertion, were analyzed by X-Gluc 115
histochemical staining in embryo, root, leaf and flower organs. None of the TET genes was 116
expressed in all organs and only a few had similar expression patterns (Fig. 1). Moreover, within 117
a specific organ, TET gene expression was not constitutive but restricted to specific domains, 118
tissues or cell types. 119
Nine TET genes, i.e. TET1, TET3, TET4, TET5, TET8, TET10, TET13, TET14 and TET15, 120
were expressed in the early globular and heart stage embryo, in which patterning takes place (Fig. 121
2). TET3 was expressed in the SAM progenitor domain of all embryo stages, TET13 in the 122
hypophysis and TET15 in the basal part of the embryo, including the hypophysis, suggesting that 123
TET3, TET13 and TET15 might participate in apical-basal patterning. TET1 was asymmetrically 124
expressed in vascular tissue precursor cells of the heart stage embryo. TET5 expression was 125
restricted to vascular tissue progenitor cells in the centre of the globular, heart and torpedo stage 126
embryo, a pattern similar to that of TMO5 (TARGET OF MONOPTEROS5), which is necessary 127
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for the periclinal cell division and vascular tissue initiation (De Rybel et al., 2013). TET4 and 128
TET10 were expressed in the central part of the embryo, including the vascular bundle progenitor 129
region; TET14 expression was restricted to the vascular progenitor strand in the cotyledons, 130
suggesting TET1, TET4, TET5, TET10 and TET14 might participate in radial patterning. TET8 131
was expressed in the apical domain of the heart stage embryo and in the tip regions of the 132
cotyledons of the torpedo and mature stage embryo (Fig. 2). After germination, expression 133
remained at the apical domains and vascular tissues, i.e. TET3 in the SAM-organizing center 134
(Supplemental Fig. S1A), TET1 and TET15 in the lateral root cap (Fig. 2), TET13 in the quiescent 135
centre (QC) of the primary root (Fig. 2), TET1, TET5, TET10 in the vascular bundle of the 136
primary root and rosette leaves (Fig. 2; Fig. 5, A and B; Supplemental Fig. S1C), and TET14 in 137
the vascular tissue of rosette leaves (Supplemental Fig. S1N). 138
Remarkably, TET2 was specifically expressed after germination in the stomatal guard cell 139
lineage during early leaf development. TET2 expression was not detected in the meristemoid 140
mother cell (MMC) that originates from a protodermal cell, but it was in the small triangle-141
shaped daughter cell (called meristemoid) generated after the first asymmetric division (Fig. 3A), 142
and not in the large daughter cell (called stomatal lineage ground cell, SLGC). TET2 expression 143
remained in the guard mother cell (GMC, Fig. 3D) after the first and subsequent asymmetric 144
divisions (Fig. 3, B and C) and in mature guard cells (Fig. 3E). 145
TET gene expression in progenitor tissues, domains and cells remained in stem cells of shoot 146
or root apical meristems or in their derived differentiated tissues or cell types in the seedling, 147
suggesting their function is required at different developmental stages. The variation in spatial 148
and temporal expression patterns of the 17 TET genes suggests a range of functions in the plant’s 149
life cycle. 150
Divergent TET Expression Patterns within Clades, but Overlapping amongst Clades 151
The TET gene family consists of different clades with duplicated or triplicated members 152
(Cnops et al., 2006; Wang et al., 2012; Boavida et al., 2013), showing diverging expression 153
profiles (Fig. 1, 2, 3). Indeed, the duplicated genes TET1 and TET2 had strikingly divergent gene 154
expression patterns. The TET1 asymmetric gene expression pattern was found in vascular tissue 155
precursor cells in the embryo, in the columella cells and vascular tissues of the primary root (Fig. 156
2), in the vascular tissues of cotyledons and rosette leaves, and in the stigma and transmitting 157
tissue of the female gametophyte (Supplemental Fig. S1, C, J and N; Supplemental Fig. S2, A and 158
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B), which correlated with tet1 mutant phenotypes observed in root and leaf (Cnops et al., 2000; 159
Cnops et al., 2006), in SAM (Chiu et al., 2007), in embryo (Lieber et al., 2011), and in fertility, 160
and which was strikingly different from TET2 expression specifically in the stomatal cell lineage 161
(Fig. 3). The duplicated genes TET3 and TET4 also had divergent expression patterns. TET3 was 162
expressed in the SAM precursor cells during embryogenesis (Fig. 2), and in the SAM-organizing 163
center of the seedling (Supplemental Fig. S1A). The TET3-GFP protein moved toward and along 164
the plasma membrane and localized itself at the plasmodesmata (Supplemental Fig. S1B; 165
Supplemental Movie S1), which confirmed previous purification from the Arabidopsis 166
Plasmodesmal Proteome (Fernandez-Calvino et al., 2011). In the primary root, TET3 was 167
expressed in the cortex, endodermis and pericycle at the differentiation zone (Fig. 2). TET4 was 168
expressed in vascular tissue progenitor cells during embryonic development, in the QC and 169
vascular tissue of the primary root (Fig. 2), in stomatal guard cells of cotyledons and anthers, and 170
in the basal region of the flower (Supplemental Fig. S1E; Supplemental Fig. S2A). Similarly, the 171
triplicated genes TET7, TET8 and TET9 had distinct expression patterns. TET7 expression was 172
restricted to the pollen (Supplemental Fig. S2C). TET8 was expressed in the apical domain of the 173
embryo, starting from the heart stage (Fig. 2), in the differentiation zone of the primary root 174
(Fig. 2), in stipules and hydathodes of rosette leaves (Supplemental Fig. S1, F and G). TET9 was 175
expressed in the vascular tissues of the primary root (Fig. 2), cotyledons and rosette leaves, in the 176
SAM, and in trichomes and surrounding pavement cells (Supplemental Fig. S1, J-L). TET11 was 177
only expressed in the pollen (Supplemental Fig. S2C), its duplicated gene, TET12, was expressed 178
in the primary root and stipules (Fig 2; Supplemental Fig. S1M), suggesting a thoroughly 179
diverged function. The triplicated genes TET13, TET14 and TET15 diverged in expression 180
patterns. TET13 was expressed in the embryonal hypophysis, in the QC, stem cells and columella 181
cells of the primary root and the LRP (Fig. 2; Fig. 4, A, E-N). TET14 expression was restricted to 182
vascular precursor cells of heart stage embryos and in the cotyledonary vascular tissues of the 183
mature embryo (Fig. 2). TET15 was expressed in the basal domain of the heart stage embryo, in 184
the LR cap and columella cells (Fig. 2), in the pollen tubes and stomatal guard cells of sepals 185
(Supplemental Fig. S2, B and C). TET16 was only expressed in the basal region of the flower and 186
pollen (Supplemental Fig. S2C), no TET17 gene expression was detected in any of the organs, 187
which was consistent with the absence of gene expression in transcriptome data of 188
Genevestigator or the eFP browser throughout development. Nevertheless, triple nuclear-189
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localized pAtTET17-GFP protein was detected in mature pollen (Boavida et al., 2013). 190
However, overlapping expression patterns were observed in TET genes of different clades in 191
pollen (TET1, TET2, TET3, TET4, TET7, TET8, TET9, TET10, TET11, TET13, TET14, TET15 192
and TET16) (Supplemental Fig. S1C) and in vascular tissues of the embryo and/or the seedling 193
(TET1, TET3, TET4, TET5, TET6, TET8, TET9, TET10, TET12 and TET14) (Fig. 2; 194
Supplemental Fig. S1), suggesting redundancy in function which might complicate functional 195
analysis by mutational approach. T-DNA insertion lines in TET genes were characterized as 196
knock-out or knock-down alleles by qRT-PCR and phenotyping in the respective mutants was 197
done with particular attention to organs in which the respective TET gene was expressed. 198
Strikingly, only in the TET13 knock-out line, tet13-1, a clear phenotype was observed in the 199
primary and lateral root development, which correlated with its site of expression (Supplemental 200
Table S1). Although the single mutant analysis was not exhaustive for putative phenotypes, the 201
identification of a phenotype for only one TET gene argued for functional redundancy amongst 202
the other TET genes and showed the need for double, triple mutant combinations. 203
TET13 Function in Primary Root Growth and Lateral Root Development 204
TET13 expression and mutant phenotypes were studied in more detail during primary and 205
lateral root development. Arabidopsis root identity is specified early during embryogenesis. The 206
cells at the basal region of the globular embryo will give rise to the hypocotyl, primary root and 207
root stem cells. The lens-shaped cells at this region are the progenitors of the QC, which is crucial 208
for maintaining root stem cells identity (van den Berg et al., 1997). In wild-type plants, the stem 209
cells surrounding the QC are maintained in the undifferentiated state. In the primary root, TET13 210
was predominately detected in the QC, most of the stem cells (cortex/endodermis initials, 211
pericycle initials, vascular tissue initials and columella initials, but not epidermis initials), the 212
first two columella cell layers and the two middle cells in the root cap (Fig. 4A). TET13 promoter 213
activity was equally present at the two QC cells of 35 seedlings, indicating that TET13 was not 214
cell cycle-regulated. The function of TET13 in primary root and LR development was studied in 215
the T-DNA insertion line tet13-1 (SALK_011012C), in which the T-DNA is located at the first 216
exon, resulting in a gene knock-out (Boavida et al., 2013). In addition, complementation of root 217
phenotypes was studied in the C1 and C5 complementation lines of tet13-1 containing one and 218
two, T-DNA loci with p35S::TET13, respectively. The primary root length was significantly 219
reduced in tet13-1 (Fig. 4, B and C), partially restored to wild type in the C1 line, and fully 220
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restored and even larger than wild type in the C5 line (Fig. 4C). The primary root length is 221
determined by several parameters such as meristem, elongation zone and differentiation zone 222
size, and cell length at these zones. The meristem size, defined as the number of cells from the 223
QC to the first elongated cell in the cortex cell file (Casamitjana-Martínez et al., 2003) was 224
significantly reduced in tet13-1 (27 ± sd 3 cells) as compared to the wild type (34 ± 4 cells) (Fig. 225
4D, Supplemental Fig. S3A). Below the QC, there are one or two layers of undifferentiated 226
columella initials that generate the differentiated columella cells containing starch that stains 227
purple by Lugol solution. One of the functions of the QC is to keep the surrounding initials in an 228
undifferentiated state because defective QC function results in a differentiated cell layer below 229
the QC instead of the columella initial cell layer (van den Berg et al., 1997, Sarkar et al., 2007). 230
Exogenous auxin (1-naphthaleneacetic acid, NAA) or auxin transport inhibitor (N-1-231
naphthylphthalamic acid, NPA) promoted the differentiation of columella initial cells in wild-type 232
seedlings, but not in mutants in auxin biosynthesis or transport (Ding and Friml, 2010). In tet13-1 233
mutants, one or two columella initial cell layers were measured in primary roots, which is 234
comparable with the wild type, and no precocious differentiation of the columella initials was 235
observed (Supplemental Fig. S3, B and C), hence, columella initial cell identity was maintained 236
in the tet13-1 mutant under normal conditions and after NAA and NPA treatment (Supplemental 237
Fig. S3, D-F). The architecture of the QC and initials was normal in the tet13-1 mutant, as 238
visualized by confocal microscopy with propidium iodide staining (Supplemental Fig. S3G), 239
indicating no function of TET13 in cytokinesis. tet13-1 seedlings were equally insensitive as 240
wild-type seedlings to hydroxyurea, a replication-blocking agent, because no dead cells were 241
observed after propidium iodide staining (Supplemental Fig. S3, H and I). 242
Auxin oscillation in the pericycle of the primary root basal meristem (De Smet et al., 2007) 243
determines the LR founder cell fate, and their anticlinal asymmetric division results in a single-244
layered primordium composed of small daughter cells flanked by two large daughter cells, termed 245
stage I. During LR development, TET13 was active at the two neighboring pericycle cells before 246
the asymmetric division (Fig. 4E). At stage I, the expression was stronger in the two newly 247
generated, small daughter cells (Fig. 4F). From stage II to VII (Fig. 4G-L), anticlinal and 248
periclinal divisions form a dome-shaped primordium with two to seven cell layers. At stage II, 249
TET13 was expressed equally strong in the tip cells at the outer layer (OL), as well as in the 250
middle two cells at the inner layer (IL) (Fig. 4G). From stage III on, TET13 expression was 251
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always stronger in the tip cells at the OL (Fig. 4, H-M) and comparable to that of the pDR5::GUS 252
reporter gene (Benková et al., 2003). At stage VIII, upon LR emergence, TET13 was expressed in 253
the QC and columella (Fig. 4N). 254
Lateral root initiation stages and LR emergence were analyzed in tet13-1 mutants: the number 255
of stage-I LRPs was severely reduced (Fig. 4O), which correlated with early TET13 expression in 256
the pericycle founder cells before the first asymmetric division (Fig. 4E); stage-IV, -V and -VI 257
LRP densities were significantly increased, whereas the number of stage-II, -III and -VII LRP 258
was slightly increased in tet13-1 (Fig. 4O); the emerged LR (EMLR) density was significantly 259
reduced (Fig. 4, O and P). Because the LRP and EMLR densities, defined as the number of LRP 260
and EMLRs per centimeter primary root, were significantly increased and reduced, respectively 261
(Fig. 4O), TET13 has a potential role in restricting LR initiation and promoting LR emergence. 262
The EMLR density in the complementation lines C1 and C5 was restored to wild type (Fig. 4P). 263
In an assay for synchronous LR initiation (Himanen et al., 2002), seedlings were first incubated 264
on medium containing the auxin transport inhibitor NPA, then transferred to NAA medium, after 265
which synchronous LR initiation was monitored. The TET13 promoter activity was induced after 266
2 h of NAA induction at the xylem pole of the pericycle, which coincides with the onset of LRI 267
(Fig. 4Q); after 3 h of NAA induction, two clear blue lines marked the pericycle (Fig. 4Q), which 268
was similar to the pDR5::GUS reporter gene expression pattern (Fig. 4R), indicating that TET13 269
expression is auxin inducible and specific for pericycle cells. In the tet13-1 mutant, the 270
pDR5::GUS reporter gene was not or very weakly expressed at the basal meristem in eight out of 271
thirteen tested seedlings (Fig. 4S), whereas in all eleven wild-type seedlings it was expressed 272
(Fig. 4T), indicating that auxin accumulation in the pericycle founder cells is affected. Hence, the 273
tet13-1 mutant allele and the complementation lines showed that TET13 has a function in the 274
primary root affecting apical meristem size and root length and in lateral root initiation and 275
emergence, which is consistent with its expression sites in the root. 276
TET5 and TET6 Have Redundant Functions in Root and Leaf Growth 277
TET5 and TET6 were the only duplicated genes with similar expression patterns, i.e. in the 278
vascular tissue of all organs, except for embryos, in which only TET5 was expressed (Fig. 1). At 279
the globular stage, TET5 expression was restricted to the vascular tissue progenitor cells (Fig. 1). 280
After germination, both TET5 and TET6 were expressed in the pericycle, the vascular tissues of 281
the primary root starting from the transition zone, the hypocotyl, cotyledons and rosette leaves 282
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(Fig. 5, A and B). Transverse sections of the primary root showed that, at the meristem-elongation 283
transition zone, TET5 and TET6 were expressed in the phloem, a few procambial cells 284
surrounding the phloem and the phloem-pole pericycle, but not in the protoxylem (Fig. 5A, lower 285
panels). At the differentiation zone, they were expressed in the pericycle and all vascular tissues 286
except for the protoxylem (Fig. 5A, upper panels). In the siliques, they were expressed in the 287
funiculus that has a function in guiding the pollen tube to reach the micropyle during fertilization 288
(Supplemental Fig. S2B). The redundant expression patterns suggest redundant functions, which 289
was confirmed by single and double mutant analyses. Several tet5 and tet6 mutant alleles were 290
analysed for their TET5 and TET6 gene expression: tet5-1 (GABI-Kat: 290A02; insert in 291
promoter), is a TET5 knock-down, tet5-2 (SALK_148216, first exon, knock-out; Boavida et al., 292
2013), tet5-3 (SALK_020009C, insert in second exon) is a TET5 knock-out, tet6-1 293
(SALK_005482C, insert in promoter), TET6 upregulated and tet6-2 (SALK_139305, insert in 294
promoter) is a TET6 knock-down (Fig. 5C). No morphological alterations or patterning defects 295
were observed in the roots, leaves or inflorescences of the single mutants tet5-1, tet5-2, tet5-3 and 296
tet6-2. However, morphological analysis of three different double mutant combinations, i.e. tet6-297
2tet5-1 (Supplemental Fig. S4), tet5-2tet6-2 (Supplemental Fig. S4) and tet5-3tet6-2 (Fig. 5E-I), 298
revealed synergistic phenotypes, such as an enlarged leaf size in seedlings at all stages of rosette 299
development (Fig. 5F) due to a significantly increased total number of cells per leaf (23,238 ± 300
2967 in tet5-3tet6-2 compared with 15,495 ± 1635 in tet5-3 and 15,197 ± 2887 in tet6-2, Fig. 301
5G), increased fresh weight in 21-d-old seedlings (Fig. 5H), longer primary roots in a root growth 302
kinetic study (Fig. 5I), confirming redundant functions of TET5 and TET6 in restricting cell 303
proliferation during root and leaf growth. 304
TET Promoter Cis-Regulatory Elements to Predict Responses to Environmental and 305
Developmental Stimuli 306
Cis-regulatory elements (or shortly motifs) were identified in the TET promoter regions using 307
an integrative bioinformatics approach (see Materials and Methods) in order to further study and 308
explain the divergence, overlap and redundancy in TET gene expression. Cis-regulatory elements 309
also provided information about environmental and developmental stimuli that might influence 310
TET gene expression, which could be used for further functional analysis. Apart from mapping 311
known cis-regulatory elements to the 2-kb promoters of the different TET genes, also co-312
regulatory genes, evolutionary sequence conservation and information about open chromatin 313
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regions (DNaseI-hypersensitive (DH) sites) were integrated to identify cis-regulatory elements 314
and TFs putatively regulating TET genes. Recently, we have shown that combining 315
complementary regulatory data sources has a positive impact on reducing the high number of 316
false positives associated with simple mapping of cis-regulatory elements and strongly increases 317
the likelihood that retained regulatory interactions are biologically functional (Vandepoele et al., 318
2009; Van de Velde et al., 2014). To identify TET cis-regulatory elements shared with co-319
regulated genes, for each TET gene, Genevestigator was first used to identify a set of tissue and 320
perturbation experiments in which the gene was strongly responsive (see Materials and Methods). 321
Next, co-regulated genes were identified under these conditions and enrichment analysis was 322
performed to identify cis-regulatory elements significantly shared between each TET gene and its 323
co-regulated genes, considering the motifs obtained by simple motif mapping, simple motif 324
mappings filtered for DH sites or filtered for evolutionary conservation (indicated with Analysis 325
type ‘motif’, ‘DH’ and ‘CM’ in Supplemental Table S2, respectively). In total, 128 cis-regulatory 326
elements were identified in the TET promoters and their respective co-regulated genes, yielding 327
regulatory information about thirteen TET genes (no result for TET11, TET13, TET16 and 328
TET17). More than half of the regulatory elements (71/128) were identified through the 329
enrichment analysis using the motif mapping files filtered for DH sites or evolutionary 330
conservation, indicating they are functionally related to accessible TF binding sites in open 331
chromatin regions or to regulatory elements that have been conserved during hundreds of 332
millions of years of evolution (Supplemental Table S2). 333
A heat map was generated with TET differential gene expression upon a selection of 334
perturbations in Genevestigator (Supplemental Fig. S5A), and used to verify the cis-regulatory 335
elements in TET promoters identified in Supplemental Table S2. Cis-regulatory elements 336
identified from TET1, TET2, TET3, TET4, TET5, TET6, TET8, TET9, TET16 correlated with their 337
expression patterns or inducible conditions. TET1 up-regulation by high light correlates with the 338
enrichment in the light-regulated gene binding sites, and induction upon brassinolide treatment 339
with a GATA PROMOTER MOTIF (Supplemental Table S2) that can be recognized by GATA 340
family TFs, some of which mediate the cross-talk between brassinosteroid and light signaling 341
pathways (Luo et al., 2010). TET2 promoter elements are mainly related to stress response, 342
including cold, dehydration, and drought response (Supplemental Table S2), and fit with the up-343
regulation of TET2 in response to abscisic acid (ABA), cold and drought (Supplemental Fig. 344
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14
S4A), which can induce stomatal closure. TET2 function in the mature stomatal guard cell might 345
relate to stomatal closure caused by stress conditions. TET3 up-regulation by ABA, cold, drought 346
and in the CBF3 overexpression background (Supplemental Fig. S4A) correlates with cold and 347
drought responsive elements and the CBFHB element that is the binding site of CBF3 (CRT/DRE 348
binding factor 3) (Supplemental Table S2), a TF involved in cold response. The TET3 expression 349
pattern in the SAM and the cold response element at the promoter region suggest a function in 350
cold sensing at the SAM or upon floral transition. TET4 is also up-regulated by ABA, cold, 351
drought and in the CBF3 overexpression background (Supplemental Fig. S4A) and correlates 352
with ABA response elements and elements in seed-specific promoters (Supplemental Table S2) 353
(Ezcurra et al., 2000; Chandrasekharan et al., 2003). TET5 and TET6, both expressed in the 354
vascular tissue, contain distinct sugar response promoter elements that suggest a role in sugar 355
sensing and growth (Supplemental Table S2). TET8 and TET9 promoter regions contain defense 356
and pathogen response elements (Supplemental Table S2), which correlate with high up-357
regulation by pathogen and elicitors (Supplemental Fig. S4A); in addition, there are three 358
endosperm-specific elements which fit with TET8 expression in the endosperm (Supplemental 359
Table S2; Supplemental Fig. S1H). One of them is the MYB98 binding site, MYB98 expression is 360
observed in the synergid cells (Kasahara et al., 2005). Presumably TET8 is expressed in the 361
synergid cells as well (Supplemental Fig. S1I). TET16 is 63 times up-regulated during pollen tube 362
growth (Supplemental Fig. S4) and it is expressed specifically in pollen (Supplemental Fig. S2C), 363
suggesting a function in this process. 364
In conclusion, the cis-regulatory element analysis confirmed the divergence in reporter 365
expression of duplicated TET genes. The presence of the same regulatory elements in TET genes 366
of different clades, such as the root-specific element in TET4 and TET10, and the vascular tissue-367
specific element in TET4, TET8, TET9 and TET12 (Supplemental Table S2), supported the 368
redundancy of the reporter expression of TET genes over the different clades. 369
TF-TET Gene Regulatory Network to Predict Function in Molecular Pathways 370
In order to obtain a better understanding of the transcriptional regulation of TETs and their 371
position in molecular pathways, a TF-TET gene regulatory network was built by combining 372
different regulatory datasets. Apart from conserved TF binding sites, also chromatin 373
immunoprecipitation (ChIP) TF binding data and TF expression perturbation information was 374
integrated to generate a TF-TET gene regulatory network (data types denoted as ‘conserved 375
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15
motif’, ‘ChIP’ and ‘DE’ in Figure 6, respectively). Although these datasets cover different 376
aspects of gene regulation, it is important to note that the obtained gene regulatory network only 377
offers a partial view on TET regulation, as for many Arabidopsis TFs, no binding site information 378
or experimental data are available. 379
The conservation of cis-regulatory elements has been used to delineate gene regulatory 380
networks in different species (Aerts, 2012) and a recent study in Arabidopsis used a phylogenetic 381
footprinting approach to delineate conserved gene regulatory networks based on known binding 382
sites for 157 TFs (Van de Velde et al., 2014). TF ChIP-Seq and ChIP-chip have been used to 383
identify target genes of different plant regulators, including TFs involved in flowering, circadian 384
rhythm and light signaling, cell cycle and hormone signaling (Mejia-Guerra et al., 2012). To 385
integrate experimental ChIP TF binding information, the network of Heyndrickx and co-workers 386
was used (Heyndrickx et al., 2014), in which 27 publicly available TF-ChIP data sets have been 387
reprocessed through an analysis pipeline consisting of quality control, platform-specific signal 388
processing, and peak calling to generate TF-TET gene interactions. Next to the physical binding 389
of a TF in the vicinity of a TET gene, which can serve as a proxy for TET gene regulation, 390
information about the regulation of a TET gene by a specific TF was also taken into account. 391
Therefore, we screened differentially expressed (DE) genes that were obtained through transcript 392
profiling after perturbation (knock-out or overexpression) of the normal activity of a TF from 15 393
publicly available studies. DE genes from five studies, in which regulatory information about 394
TET genes was reported, were retained, complementing the ChIP binding data for AGL15, BES1, 395
LFY and FUS3 (see Materials and Methods). Most of the TETs were regulated by multiple TFs, 396
i.e. TET3, TET4, TET8, TET9 and TET14. Some duplicated TETs shared common TFs, i.e. TET3 397
and TET4, TET5 and TET6, TET8 and TET9 (Fig. 6). 398
To further validate regulatory interactions in the network, such as those inferred through ChIP 399
and conserved TF binding sites, expression perturbation data from Genevestigator was obtained 400
for nine TFs (AGL15, AP1, PI, EIN3, LFY, PIF4, PIF5, TOC1 and SR1) and TFs-TETs 401
interactions were confirmed by significant differential expression after TF perturbation (Table 1). 402
The TFs in the regulatory networks were related to light response (PIF4, PIF5), circadian clock 403
(TOC1), floral organ identity (AP1, AP2, AP3, SEP3, PI), flowering initiation (AGL15, LFY) 404
and defence response (EIN3, MYB4, SR1) and identify the respective TET genes as novel 405
components of specific developmental or physiological pathways. 406
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16
DISCUSSION 407
Arabidopsis TET genes are expressed in different organs/tissues and cell types during 408
embryonic and vegetative development. Intriguingly, the onset of expression coincided with the 409
onset of patterning and cell specification at globular or heart stage embryos (TET1, TET3, TET4, 410
TET5, TET8, TET10, TET13, TET14 and TET15) and in seedlings at the initiation of the stomatal 411
cell lineage (TET2), or at the asymmetric division in the primary root pericycle upon lateral root 412
initiation (TET13), which suggests a role for these TET genes in cell specification. Plant cells are 413
surrounded by a rigid, but dynamic, cell wall, which prevents them from migration during 414
specification, hence, they acquire their fate by positional information from neighboring cells. The 415
cellular communication involves diffusible or actively transported molecules, such as peptides, 416
small RNAs, transcription factors or phytohormones, that upon recognition by competent cells 417
trigger signaling cascades that result in the initiation or repression of specific developmental 418
programs (Sparks et al., 2013). Remarkably, nine TETRASPANINs (TET1, TET3, TET4, TET5, 419
TET8, TET10, TET13, TET14 and TET15) are expressed in embryonic progenitor tissue and three 420
early in cell lineage of the seedling until differentiation (TET2, TET9 and TET13). The expression 421
in the progenitor cells of a cell lineage suggests they might be required in progenitor cell identity 422
establishment or maintenance, or in lateral inhibition of neighboring cells. Their expression later 423
in the differentiated cells of the same cell lineage, suggests a function in the physiology or 424
biochemistry of the mature cells, which might be related or not to its early function. In animals, 425
the same tetraspanin can be expressed in a cell lineage and is required for the expression of the 426
partner protein, whereas the partner protein is expressed more stage-specifically (Shoham et al., 427
2003). Hence, the expression of certain TET genes in plants, very early in a cell lineage until 428
differentiated state, might indicate dynamic interactions in time with partner proteins that are 429
components of different molecular pathways. 430
Most of the duplicated TET genes have diverged expression patterns during the plant’s life 431
cycle, as was demonstrated in the pTET-reporter lines and coincided with different regulatory 432
elements in their promoters (Supplemental Table S2). Even though the duplicated TET5 and 433
TET6 genes showed redundant expression in vascular tissue, different regulatory elements related 434
to sugar sensing were identified in their promoters (Supplemental Table S2). Largely overlapping 435
expression patterns were observed in TET genes over the clades in embryos, primary roots, 436
rosette leaves and pollen, and might refer to the conservation of ancestral TET gene functions in 437
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17
specific tissues or cells. Indeed, similar regulatory elements were identified in TET promoters 438
belonging to different clades such as ABA-responsive, root-specific and light-related motifs, 439
which add to putative overlap in function (Supplemental Table S2). So, here, it might be more 440
logical to suggest these paralogous TET genes evolved in their promoter, through sub-441
functionalization (Moore and Purugganan, 2005). 442
These overlaps might explain the mild or undetectable phenotypes in single mutants in our 443
mutational analysis (Supplemental Table S1), and demonstrates the need for making double or 444
triple mutant combinations over the clades to detect phenotypes. Such a strategy was rewarding 445
in animals, resulting in strong phenotypes in mice and Drosophila melanogaster (Fradkin et al., 446
2002; Rubinstein et al., 2006). 447
TET5 and TET6 duplicated genes have the same promoter activity pattern in post-embryonic 448
vascular tissues and in cells surrounding procambial cells and the phloem, suggesting a putative 449
redundant function in vascular bundle activity, such as in nutrient or photoassimilates transport, 450
rather than in patterning, because no venation patterning defect was observed in the double 451
tet5tet6 mutant. Combining the single tet5 and tet6 mutants resulted in a significantly enhanced 452
growth by cell proliferation in root and shoot, suggesting that TET5 and TET6 restrict growth 453
through a sugar-sensing mechanism because such cis-regulatory elements were identified in their 454
promoters (Supplemental Table S2). Indeed, sucrose promotes cell proliferation by activating cell 455
cycle (Riou-Khamlichi et al., 2000). Arabidopsis tetraspanins can form homo- and heterodimers 456
when expressed in yeast (Boavida et al., 2013). The redundant function of TET5 and TET6 457
suggests that for proper activity, heterodimers might need to be formed or interaction with a 458
common protein is required, which needs further testing. 459
TET13 expression in the pericycle founder cells and the gradual spatial pattern in the LRP 460
resembles auxin accumulation and gradients that regulate LR development (Benková et al., 461
2003). TET13 shows overlapping expression in the pericycle with TET3, TET4, TET5, TET6, 462
TET8, TET9, TET10 and TET12, that might explain the mild phenotype in LR development in 463
tet13-1. Significant reduction of stage-I LRP density in tet13-1 indicates that LR initiation is 464
affected and TET13 is required to promote LR initiation. It nicely correlated with TET13-specific 465
expression in the two pericycle founder cells before the asymmetric divisions that precede LR 466
initiation and with its early inducible expression in the xylem-pole pericycle at the root basal 467
meristem upon NAA treatment (Himanen et al., 2002). Significant increase of stage-II to -VII 468
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LRP densities but reduced EMLR density indicate that the emergence of the LR is also affected 469
in tet13-1, a process that is highly regulated by the transcellular auxin-dependent signaling 470
pathway that results in cell wall remodeling in the adjacent endodermal, cortical and epidermal 471
cells overlaying the primordium (Swarup et al., 2008). Thus, TET13 regulates two different 472
aspects of LR development, restricting LR initiation and promoting LR emergence. 473
Meta-analysis of gene responses to certain perturbations gives a general idea about the gene 474
function in biological processes. However, these responses, as measured through differential 475
expression, could be due to the indirect or secondary regulation. The regulatory elements residing 476
in the promoter regions are primarily responsible for the gene response to the perturbations, 477
because they are the binding sites for the TFs that regulate gene expression. The identification of 478
upstream TFs that bind to these regulatory elements together with other regulatory data sets 479
complemented the inference of TET gene functions and allowed to position TET genes in specific 480
regulatory networks describing flowering time, circadian clock and defense response. Integration 481
of the different expression and regulatory analyses presented in this work suggests function for 482
TET3 in flowering response under low temperature. Indeed, TET3 is expressed in the SAM-483
organizing center progenitor cells in embryos that remain in the seedling. Cold and dehydration 484
response elements are enriched in the TET3 promoter that correlate with up-regulation of TET3 485
gene expression by ABA, cold and drought treatment. The PRR5, AGL15, and PIF4 TFs that 486
regulate TET3 (Fig. 6) have a function in flowering response (Adamczyk et al., 2007; Nakamichi 487
et al., 2007; Kumar et al., 2012), and in the TF genetic perturbation backgrounds, AGL15 and 488
LFY expression levels are negatively correlated with TET3 (Table 1). Thus, the meta-analysis is 489
hypothesis-generating and will be helpful for experimental design in further functional analyses. 490
Our analyses suggest a function for TET8 in defense response. The TET8 gene is up-regulated 491
upon pathogen treatment, which correlated with enrichment of defense response cis-regulatory 492
elements in its promoter. The SR1/CAMTA3, MYB4 and EIN3 TFs that putatively regulate 493
TET8 (Fig. 6) are involved in defense response; SR1 suppresses defense response (Nie et al., 494
2012), MYB4 is a transcriptional repressor that is induced by elicitor FLG22 (Schenke et al., 495
2011), and EIN3 activates downstream immune-responsive genes in an ethylene-dependent way, 496
such as FLS2, which has EIN3 binding sites at the promoter region (Boutrot et al., 2010). TET8 is 497
up-regulated in the loss-of-function sr1 allele and down-regulated in the ein3-1eil1-1 double 498
mutant, indicating that TET8 expression is antagonistically regulated by SR1 and EIN3 in the 499
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19
immune signaling pathway. TET8 gene expression was significantly induced by the FLG22 and 500
EF-Tu elicitors that mimicked pathogen infection (Supplemental Fig. S4B), which confirmed the 501
perturbation heat map, the cis-regulatory elements in the promoter region and the TF-TET8 gene 502
regulatory network. 503
In conclusion, combining in planta expression and mutational analyses with meta-analyses on 504
perturbation responses, cis-regulatory element identification, and the construction of a TF-TET 505
regulatory network provided an exciting novel and integrated approach, revealing already the 506
function of a few plant tetraspanins and enabling the functional prediction of many more TET 507
genes in future work. 508
MATERIALS AND METHODS 509
Plant Materials and Growth Conditions 510
The mutant lines tet5-1 (GABI-Kat: 290A02), tet5-2 (SALK_148216), tet5-3 511
(SALK_020009C), tet6-1 (SALK_005482C), tet6-2 (SALK_139305) and tet13-1 512
(SALK_011012C) were obtained from Arabidopsis GABI-Kat (http://www.gabi-kat.de/) and the 513
European Arabidopsis Stock Centre (http://www.arabidopsis.org/), respectively. The presence of 514
the T-DNA was confirmed by PCR with a T-DNA-specific and gene-specific primers 515
(Supplemental Table S3). 516
Seeds were germinated on Murashige and Skoog medium supplemented with 1% (w/v) 517
sucrose, 0.8% (w/v) agarose, pH 5.7. Seeds were surface sterilized and stratified at 4°C for two 518
nights and moved to the growth chamber. For pAtTET::NLS-GFP/GUS lines, 6-day-old seedlings 519
grown vertically at 21°C under 24-h light conditions (75~100 µmol m-2 s-1) were used for SAM 520
and root analysis; 14-day-old seedlings corresponding to the growth stage 1.04 (Boyes et al., 521
2001) and grown horizontally at 21°C under 16-h light/ 8-h dark conditions were used for 522
cotyledon, emerging leaf 1, 2, 3 and 4 primordia analysis; 6-w-old plants grown in the soil at 523
21°C under 16-h light/ 8-h dark conditions were used for analysis of inflorescences, different 524
stages of flowers, siliques and embryos. 525
Promoter and Open Reading Frame Cloning, Construction of Expression Vectors and Plant 526
Transformation 527
Promoter sequences, defined as intergenic regions with sizes ranging between 396 and 3400 528
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20
bp, and open reading frame sequences were amplified from the Arabidopsis thaliana genome 529
with primers listed in Supplemental Table S2 and cloned into the entry vectors using BP clonase 530
(Invitrogen) to generate the entry clone. 531
For complementation of tet13-1, full-length genomic DNA of AtTET13 was amplified with 532
primers listed in Supplemental Table S3 and cloned into the entry vectors using BP clonase 533
(Invitrogen) to generate the entry clone. The expression clones were constructed by LR clonase 534
(Invitrogen) with the entry clone and the destination vectors pMK7S*NFm14GW (pAtTET1-535
17::NLS-GFP/GUS), pK7FWG2 (p35S::TET3:GFP) and pB2GW7 (p35S::TET13) (Karimi et al., 536
2007). The plasmids were transferred into Agrobacterium tumefaciens pMP90 cells. All 537
constructs were transferred into Arabidopsis ecotype Columbia-0 (Col-0) or tet13-1 by floral dip 538
transformation. 25 mg transgenic seeds of the T1 generation was used for high-density plating on 539
50 mg/L kanamycin (pAtTET1-17::NLS-GFP/GUS and p35S::TET3:GFP) or 7.5 mg/L DL-540
phosphinothricin (Duchefa) (complementation of tet13-1 with p35S::TET13). The resistant 541
seedlings were transferred into soil for T2 generation seed harvest. 542
For pAtTET1-17::NLS-GFP/GUS reporter lines, the number of T-DNA loci was analyzed in 7 543
to 35 T2 populations per construct after germination on kanamycin, three lines per construct with 544
a single-locus insertion were analyzed by X-Gluc histochemical staining to score the promoter 545
activity during development. For the complementation analysis of tet13-1, T2 seedlings of lines 546
C1 and C5 containing one and two T-DNA loci with p35S::TET13, respectively, were genotyped 547
for the presence of the p35S::TET13 T-DNA using bar primers (Supplemental Table S3), and 548
used for primary root growth and emerged lateral root density measurements. 549
Histochemical, Histological and Phenotypic Analyses and Statistical Tests 550
The 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc) assay was performed as described 551
previously (Coussens et al., 2012). Ovules were cleared overnight in an 8: 2: 1 (g: mL: mL) 552
mixture of chloral hydrate: distilled water: glycerol and mounted on the microscope slides with 553
the same solution (Grini et al., 2002). For the LRP staging and a better visualization of the TET13 554
expression pattern in the LRP, the samples were treated as described previously with some 555
modifications (Malamy and Benfey, 1997). Both fresh and stained samples were fixed in 70% 556
ethanol overnight and transferred into 4% HCl, 20% methanol and incubated at 62°C for 40 min 557
in 7% NaOH and 60% ethanol at room temperature for 15 minutes. The samples were then 558
rehydrated for 10 min in either 60%, 40%, 20% or 10% ethanol at room temperature. Finally, the 559
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21
samples were infiltrated in 25% glycerol and 5% ethanol for 10 min and mounted with 50% 560
glycerol. The root meristem size was determined as the number of cells in the cortex cell file 561
from the QC to the first elongated cell (Casamitjana-Martínez et al., 2003). The samples were 562
mounted with 50% lactic acid and observed immediately. For the hydroxyurea, NAA and NPA 563
treatment, 5-d-old seedlings were transferred onto the MS medium supplemented for 24 h with 564
1 mM hydroxyurea, 1 µM NAA or 1 µM NPA, respectively. Seedlings treated with hydroxyurea 565
were counterstained with propidium iodide (PI) and observed with a confocal microscope. 566
Seedlings treated with NAA or NPA were also treated for 1 min with Lugol solution to stain the 567
starch granules, mounted with chloral hydrate and checked immediately with microscope. 568
For the epidermal cell imaging, leaves were fixed in 100% ethanol overnight and mounted with 569
90% lactic acid. The leaf area was measured with the ImageJ software (http://rsbweb.nih.gov/ij/). 570
The epidermal cells on the abaxial side were drawn with a DMLB microscope equipped with a 571
drawing tube and differential interference contrast objectives. The total number of cells per leaf 572
was determined as described previously (De Veylder et al., 2001). 573
All samples were imaged with a binocular Leica microscope or an Olympus DIC-BX51 574
microscope. The confocal images were taken with an Olympus Fluo View FV1000 microscope or 575
Zeiss LSM5 Exiter confocal. The fluorescence was detected after a 488 nm (GFP), 543 nm (PI) 576
or 514 nm (FM4-64) excitation and an emission of 495-520 nm for GFP, 590-620 nm for PI and 577
600-700 nm for FM4-64. Transverse section was done according to De Smet et al. (2004). 578
Means between samples were compared by a two-tailed Student’s t-test, an f-test was assessed 579
for the equality between population variances. 580
Elicitor Treatment 581
EF-Tu and FLG22 were synthesized and purchased from GenScript 582
(http://www.genscript.com). Col-0 plant samples were grown on MS medium for 16 days and 583
transferred into liquid MS medium supplemented with EF-Tu or FLG22 at the final concentration 584
of 1 µM for 2 h on an orbital shaker. 585
Quantitative Real-Time PCR Analysis 586
Total RNA was prepared with the RNeasy kit (QIAGEN). One µg RNA was used as template 587
to synthesize cDNA with the iScript cDNA synthesis Kit (Bio-Rad). The expression level was 588
analyzed on a LightCycler 480 apparatus (Roche) with SYBR Green and all reactions were 589
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22
performed in three technical replicates. Expression levels were normalized to reference genes 590
PP2A and UBC. 591
Cis-Regulatory Element Analysis 592
Known TF binding sites and DNA motifs were mapped on the 2-kb upstream sequence for all 593
genes and for all included species using DNA-pattern allowing no mismatches (Thomas-Chollier 594
et al., 2008). 692 cis-regulatory elements were obtained from AGRIS (Davuluri et al., 2003), 595
PLACE (Higo et al., 1999) and Athamap (Steffens et al., 2004). In addition, 44 positional count 596
matrices were obtained from Athamap and for 15 TFs, positional count matrices were obtained 597
from ChIP-Seq data (Heyndrickx et al., 2014). Positional count matrices were mapped genome-598
wide using MatrixScan using a p-value cut-off <1e-05 (Thomas-Chollier et al., 2008). In order to 599
reduce the high false-positive rates associated with interfering regulatory interactions based on 600
simple motif mapping, two types of filtering were used. A first approach to enrich for functional 601
interactions consisted of filtering motif matches using cross-species sequence conservation or 602
open chromatin regions. Therefore, motif matches were filtered using conserved non-coding 603
sequences (Van de Velde et al., 2014) and DNaseI hypersensitive (DH) sites. The DH sites were 604
downloaded from the NCBI SRA database (accession ID SRP009678) (Zhang et al., 2012). A 605
second approach started from tightly co-regulated genes to identify co-regulatory motifs or TF 606
binding sites. For each TET gene, motif mapping information was combined with a set of co-607
regulated genes from Genevestigator. In the “perturbations tool”, the perturbations under which 608
TET genes were two times up-regulated (p-value < 0.05) were selected to create new sample sets. 609
Then, in the “co-expression tool”, the top 200 positively correlated genes under the categories 610
“anatomy” and “perturbations” were selected. The overlapping genes were defined as “co-611
regulated” genes. Only motifs present in the upstream or downstream TET sequence and showing 612
significant enrichment in the co-regulation cluster were retained. Motif enrichment was 613
determined using the hypergeometric distribution using false discovery rate correction. Only 614
significantly (p-value < 0.05) enriched motifs were retained. 615
Gene Regulatory Network Inference 616
The gene regulatory networks based on conserved non-coding sequences from Van de Velde et 617
al. (2014) and on TF-ChIP data from Heyndrickx et al. (2014) were filtered for TET target genes. 618
For TFs with ChIP data, DE genes after TF perturbation were also included for AT5G13790 619
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23
(AGL15) (Zheng et al., 2009), AT1G19350 (BES1) (Yu et al., 2011), AT5G61850 (LFY) (Schmid 620
et al., 2003) and AT3G26790 (FUS3) (Yamamoto et al., 2010; Lumba et al., 2012). For other TFs, 621
DE genes after TF perturbation were obtained through Genevestigator. 622
GO Enrichment 623
GO enrichment was performed using the hypergeometric distribution with Bonferroni 624
correction. 625
Supplemental Data 626
The following supplemental materials are available. 627
Supplemental Figure S1. TET expression in seedlings, seeds and TET3 protein localization. 628
Supplemental Figure S2. TET expression in flower organs. 629
Supplemental Figure S3. tet13-1 primary root morphology. 630
Supplemental Figure S4. tet5 and tet6 single and double mutant phenotypes in other alleles 631
Supplemental Figure S5. Heat map of TET response to different perturbations and TET8 632
expression levels after elicitors treatment. 633
Supplemental Table S1. tet mutants collected in this study. 634
Supplemental Table S2. TET cis-regulatory element information. 635
Supplemental Table S3. Primers used in the study. 636
Supplemental Movie S1. TET3-GFP movement on the plasma membrane. 637
ACKNOWLEDGMENTS 638
The authors would like to thank Annick Bleys for help in preparing the manuscript, Matyas 639
Fendrych for help in confocal imaging, Carina Braeckman for plant transformation. 640
641
AUTHOR CONTRIBUTIONS 642
FW designed, performed and interpreted experiments, wrote the manuscript; AM, PN performed 643
experiments, JVdV and KVDP designed, performed and interpreted the bioinformatic analyses, 644
wrote the manuscript; KH designed and interpreted experiments; MVL designed the project and 645
experimental approach, coordinated the work, wrote the manuscript. 646
647
648
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24
FIGURE LEGENDS 649
650
Figure 1. Schematic overview of TET expression patterns in different organs. Duplicated gene 651
pairs are indicated with square brackets (The length of the square brackets does not represent the 652
evolutionary distance). E: embryo; R: root; m: primary root meristem and root tip; d: primary 653
root differentiation zone; lrp: lateral root primordia; C: cotyledon; L: rosette leaf; F: flower; se: 654
sepal; pe: petal; st: stamen; ca: carpel. 655
656
Figure 2. TET expression patterns during embryogenesis and in the primary root meristem after 657
germination. Transgenic plants shown are: pAtTET1::NLS-GFP/GUS, pAtTET3::NLS-GFP/GUS, 658
pAtTET4::NLS-GFP/GUS, pAtTET5::NLS-GFP/GUS, pAtTET8::NLS-GFP/GUS, 659
pAtTET10::NLS-GFP/GUS, pAtTET13::NLS-GFP/GUS, pAtTET14::NLS-GFP/GUS, and 660
pAtTET15::NLS-GFP/GUS. Columns represent consecutive developmental stages: globular, 661
heart, torpedo and mature. Dotted lines delineate the outlines of the young embryos. Arrows in 662
TET1, TET3 and TET14 panels indicate gene expression sites. Scale bars represent 0.01 mm 663
(globular, heart and torpedo stages), 0.1 mm (mature embryo and root meristem). 664
665
Figure 3. Diagram of Arabidopsis stomatal development (left) and the respective TET2 666
expression patterns at the abaxial epidermis of 6-d-old cotyledons of the pAtTET2::NLS-667
GFP/GUS line (right). A, TET2 expression in the meristemoid after entry division. B and C, 668
TET2 expression in the meristemoid after one or two additional asymmetric divisions. The arrow 669
indicates the meristemoid. D and E, TET2 expression in the GMC and the mature guard cells, 670
respectively. Cells in different colors are: MMC, gray; meristemoid, blue; SLGC, white; GMC, 671
red; guard cell, green; pavement cell, light green. Scale bars represent 0.01 mm. 672
673
Figure 4. TET13 expression in the primary root and LRP, tet13-1 root phenotypes and 674
complementation. Transgenic plants shown are: pAtTET13::NLS-GFP/GUS (A, E-N and Q), 675
tet13-1 (B, C, D, O, P)C1 and C5 (tet13-1 containing one, respectively two T-DNA loci with 676
p35S::TET13) (C, P), pDR5::GUS (R and T) and tet13-1xpDR5::GUS (S). A, TET13 expression 677
in the primary root tip. B, tet13-1 scheme, 12-d-old seedlings of Col-0 and tet13-1 growing 678
vertically under 24-h light condition. C, Primary root growth kinetics. Means are presented ± sd 679
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25
(n=22-33). Asterisks mark significant differences: *p < 0.05, **p < 0.01 and ***p < 0.001. D, 680
Root meristem size (defined as the number of RM cells) of 6-d-old seedlings. Means are 681
presented ± sd (n=28-39). E-N, Detailed expression analysis of TET13 at different stages during 682
LR development. Corresponding developmental stages I-VII and EMLR are indicated in the top 683
right corner. IL and OL: inner and outer layers, respectively; ep: epidermis; c: cortex; en: 684
endodermis; p: pericycle. Arrowheads indicate the division planes. Inset in I shows the top view 685
of stage IV. O, Staging of LRP and EMLR densities. Means are presented ± sd (n=20). P, EMLR 686
densities. Means are presented ± sd (n=22-33). ns, not significant. Asterisks mark significant 687
differences: *p < 0.05, **p < 0.01 and ***p < 0.001. Q and R, NAA-induced TET13 (Q) and 688
pDR5::GUS (R) expression. The seedlings were grown on 10 µM NPA medium for 72 h after 689
germination and then transferred to 10 µM NAA medium for 0 h, 2 h and 3 h. S and T, 690
DR5::GUS expression patterns at the root basal meristem in 7-d-old tet13-1xpDR5::GUS (S) and 691
wild-type (T) seedlings. Q-T, black arrowheads indicate sites of inducible expression. Scale bars 692
represent 0.01 mm (E-N), 0.1 mm (S and T), 0.5 mm (Q and R). 693
694
Figure 5. TET5 and TET6 expression, and tet5-3, tet6-2 and tet5-3tet6-2 mutant phenotypes. 695
Transgenic plants shown are: pAtTET5::NLS-GFP/GUS, pAtTET6::NLS-GFP/GUS, tet5-3, tet6-2 696
and tet5-3tet6-2. A, TET5 and TET6 expression in the vascular tissue of the meristem-elongation 697
transition zone (between dotted lines) and the differentiation zone (upper) of the primary root. 698
Transverse sections show the transition zone and differentiation zone, as indicated by the dashed 699
lines. ep: epidermis; c: cortex; e: endodermis; p: pericycle. Arrowheads and arrows indicate 700
phloem-pole, pericycle and protoxylem, respectively. B, TET5 and TET6 expression in the rosette 701
leaves. C, TET5 and TET6 schemes with T-DNA insertions. Bold, thin and breaking lines indicate 702
exons, introns and promoter, respectively. D, Relative TET5 (right) and TET6 (left) transcript 703
level in 7-d-old seedlings measured by qRT-PCR. Means are presented ± sd (n=3). E, Rosette and 704
leaf series of 21-d-old seedlings. From left to right are leaf 1 to leaf 8, incisions were made to 705
make the leaves fully expanded when necessary. F, Quantification of rosette leaf size in E. Means 706
are presented ± sd (n=8-10). G, Total number of cells per leaf. Leaf 1 and/or 2 were used. Means 707
are presented ± sd (n=3). H, Fresh weight of 21-d-old seedlings. Only rosette leaves were used 708
for the experiment. I, Primary root growth kinetics (root length). Means are presented ± sd (n=31-709
39). Asterisks in all graphs mark significant differences: *p < 0.05, **p < 0.01 and ***p < 0.001. 710
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Scale bars represent 0.01 mm (A, transverse section), 0.1 mm (A, longitudinal section), and 1 mm 711
(B). 712
713
Figure 6. TF-TET gene regulatory network. Nodes and arrows depict genes and regulatory 714
interactions, yellow and gray rounded rectangles are TFs and TETs, respectively. The arrows do 715
not represent any positive or negative regulation between the TFs and TETs. Regulation identified 716
by ChIP-Seq and conserved binding motif are shown with doubled lines and solid lines, 717
respectively. Differentially expressed regulation is shown with dashed lines. AGL15: 718
AGAMOUS-LIKE 15, AP1: APETALA1, AP2: APETALA2, AP3: APETALA3, ATAF2: 719
ARABIDOPSIS NAC DOMAIN CONTAINING PROTEIN 81, BES1: BRI1-EMS-720
SUPPRESSOR 1, EIN3: ETHYLENE-INSENSITIVE 3, ERF115: ETHYLENE RESPONSE 721
FACTOR 115, FHY3: FAR-RED ELONGATED HYPOCOTYLS 3, GL3: GLABRA3, LFY: 722
LEAFY, MYB4: MYB DOMAIN PROTEIN 4, PI: PISTILLATA, PIF4: PHYTOCHROME 723
INTERACTING FACTOR 4, PIF5: PHYTOCHROME INTERACTING FACTOR 5, PRR5: 724
PSEUDO-RESPONSE REGULATOR 5, SEP3: SEPALLATA3, SR1/CAMTA3: SIGNAL 725
RESPONSIVE 1/CALMODULIN-BINDING TRANSCRIPTION ACTIVATOR 3, TOC1: 726
TIMING OF CAB EXPRESSION 1. 727
728
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TABLE 729
Table 1. TETs transcript changes upon TF perturbation. Genevestigator microarray studies on 730
genotypes perturbed for the TFs identified in Figure 5 were analyzed for differential TET 731
transcript levels at p-value < 0.05. In double mutant and amiRNA backgrounds, only the TFs of 732
interest were listed. Fold-change was calculated from Log(2)-ratio, DE, differentially expressed. 733
"-" and "+" represent down-regulation and up-regulation, respectively. *, data were obtained from 734
the same studies in Figure 6. 735
Genotype DE TF fold-change DE TF p-value DE TET fold-change
DE TET p-value
agl15-4agl18-1/Col* AGL15 -3.77 <0.001
TET3 +1.35 0.03 TET4 +1.66 <0.001 TET5 +1.17 0.207 TET6 +1.33 0.039 TET8 +1.80 0.019 TET9 +2.17 <0.001 TET11 +1.18 0.107
35S::amiR-mads-2(MIR319a)_strong/Col-0
AP1 -5.24 <0.001 TET4 -1.18 0.051 TET14 -1.04 0.71
PI -1.95 0.004 TET3 -1.77 0.054 TET4 -1.18 0.051 TET14 -1.04 0.71
ein3-1eil1-1/coi1-2 EIN3 -9.53 0.003 TET8 -1.40 0.049 35S:LFY-GR/Ler LFY +36.58 <0.001 TET3 -1.19 0.002 lfy-12/Col-0* (shoot apex, rosette is 50% of final size) LFY -3.33 0.007 TET3 +1.63 0.006
lfy-12/Col-0* (inflorescence, first flower open) LFY -1.82 0.006 TET3 +1.80 0.018
pif4-101pif5-3/Col-0 PIF4, PIF5 -1.04, -70.1 0.746, <0.001
TET3 -1.28 0.008 TET14 +1.24 0.064
Alc::TOC1/Col-0 TOC1 +8.59 <0.001 TET8 -1.17 0.013 camta3-2/Col-0 SR1 -5.61 0.003 TET8 +1.88 0.027
736
737
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Figure 1. Schematic overview of TET expression patterns in different organs. Duplicated
gene pairs are indicated with square brackets (The length of the square brackets does not
represent the evolutionary distance). E: embryo; R: root; m: primary root meristem and root
tip; d: primary root differentiation zone; lrp: lateral root primordia; C: cotyledon; L: rosette
leaf; F: flower; se: sepal; pe: petal; st: stamen; ca: carpel.
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Figure 2. TET expression patterns during embryogenesis and in the primary root meristem
after germination. Transgenic plants shown are: pAtTET1::NLS-GFP/GUS, pAtTET3::NLS-
GFP/GUS, pAtTET4::NLS-GFP/GUS, pAtTET5::NLS-GFP/GUS, pAtTET8::NLS-GFP/GUS,
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pAtTET10::NLS-GFP/GUS, pAtTET13::NLS-GFP/GUS, pAtTET14::NLS-GFP/GUS, and
pAtTET15::NLS-GFP/GUS. Columns represent consecutive developmental stages: globular,
heart, torpedo and mature. Dotted lines delineate the outlines of the young embryos. Arrows
in TET1, TET3 and TET14 panels indicate gene expression sites. Scale bars represent
0.01 mm (globular, heart and torpedo stages), 0.1 mm (mature embryo and root meristem).
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Figure 3. Diagram of Arabidopsis stomatal development (left) and the respective TET2
expression patterns at the abaxial epidermis of 6-d-old cotyledons of the pAtTET2::NLS-
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GFP/GUS line (right). A, TET2 expression in the meristemoid after entry division. B and C,
TET2 expression in the meristemoid after one or two additional asymmetric divisions. The
arrow indicates the meristemoid. D and E, TET2 expression in the GMC and the mature guard
cells, respectively. Cells in different colors are: MMC, gray; meristemoid, blue; SLGC, white;
GMC, red; guard cell, green; pavement cell, light green. Scale bars represent 0.01 mm.
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Figure 4. TET13 expression in the primary root and LRP, tet13-1 root phenotypes and
complementation. Transgenic plants shown are: pAtTET13::NLS-GFP/GUS (A, E-N and Q),
tet13-1 (B, C, D, O, P)C1 and C5 (tet13-1 containing one, respectively two T-DNA loci with
p35S::TET13) (C, P), pDR5::GUS (R and T) and tet13-1xpDR5::GUS (S). A, TET13
expression in the primary root tip. B, tet13-1 scheme, 12-d-old seedlings of Col-0 and tet13-1
growing vertically under 24-h light condition. C, Primary root growth kinetics. Means are
presented ± sd (n=22-33). Asterisks mark significant differences: *p < 0.05, **p < 0.01 and
***p < 0.001. D, Root meristem size (defined as the number of RM cells) of 6-d-old
seedlings. Means are presented ± sd (n=28-39). E-N, Detailed expression analysis of TET13 at
different stages during LR development. Corresponding developmental stages I-VII and
EMLR are indicated in the top right corner. IL and OL: inner and outer layers, respectively;
ep: epidermis; c: cortex; en: endodermis; p: pericycle. Arrowheads indicate the division
planes. Inset in I shows the top view of stage IV. O, Staging of LRP and EMLR densities.
Means are presented ± sd (n=20). P, EMLR densities. Means are presented ± sd (n=22-33).
ns, not significant. Asterisks mark significant differences: *p < 0.05, **p < 0.01 and ***p <
0.001. Q and R, NAA-induced TET13 (Q) and pDR5::GUS (R) expression. The seedlings
were grown on 10 µM NPA medium for 72 h after germination and then transferred to 10 µM
NAA medium for 0 h, 2 h and 3 h. S and T, DR5::GUS expression patterns at the root basal
meristem in 7-d-old tet13-1xpDR5::GUS (S) and wild-type (T) seedlings. Q-T, black
arrowheads indicate sites of inducible expression. Scale bars represent 0.01 mm (E-N),
0.1 mm (S and T), 0.5 mm (Q and R).
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Figure 5. TET5 and TET6 expression, and tet5-3, tet6-2 and tet5-3tet6-2 mutant phenotypes.
Transgenic plants shown are: pAtTET5::NLS-GFP/GUS, pAtTET6::NLS-GFP/GUS, tet5-3,
tet6-2 and tet5-3tet6-2. A, TET5 and TET6 expression in the vascular tissue of the meristem-
elongation transition zone (between dotted lines) and the differentiation zone (upper) of the
primary root. Transverse sections show the transition zone and differentiation zone, as
indicated by the dashed lines. ep: epidermis; c: cortex; e: endodermis; p: pericycle.
Arrowheads and arrows indicate phloem-pole, pericycle and protoxylem, respectively. B,
TET5 and TET6 expression in the rosette leaves. C, TET5 and TET6 schemes with T-DNA
insertions. Bold, thin and breaking lines indicate exons, introns and promoter, respectively. D,
Relative TET5 (right) and TET6 (left) transcript level in 7-d-old seedlings measured by qRT-
PCR. Means are presented ± sd (n=3). E, Rosette and leaf series of 21-d-old seedlings. From
left to right are leaf 1 to leaf 8, incisions were made to make the leaves fully expanded when
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necessary. F, Quantification of rosette leaf size in E. Means are presented ± sd (n=8-10). G,
Total number of cells per leaf. Leaf 1 and/or 2 were used. Means are presented ± sd (n=3). H,
Fresh weight of 21-d-old seedlings. Only rosette leaves were used for the experiment. I,
Primary root growth kinetics (root length). Means are presented ± sd (n=31-39). Asterisks in
all graphs mark significant differences: *p < 0.05, **p < 0.01 and ***p < 0.001. Scale bars
represent 0.01 mm (A, transverse section), 0.1 mm (A, longitudinal section), and 1 mm (B).
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Figure 6. TF-TET gene regulatory network. Nodes and arrows depict genes and regulatory
interactions, yellow and gray rounded rectangles are TFs and TETs, respectively. The arrows
do not represent any positive or negative regulation between the TFs and TETs. Regulation
identified by ChIP-Seq and conserved binding motif are shown with doubled lines and solid
lines, respectively. Differentially expressed regulation is shown with dashed lines. AGL15:
AGAMOUS-LIKE 15, AP1: APETALA1, AP2: APETALA2, AP3: APETALA3, ATAF2:
ARABIDOPSIS NAC DOMAIN CONTAINING PROTEIN 81, BES1: BRI1-EMS-
SUPPRESSOR 1, EIN3: ETHYLENE-INSENSITIVE 3, ERF115: ETHYLENE RESPONSE
FACTOR 115, FHY3: FAR-RED ELONGATED HYPOCOTYLS 3, GL3: GLABRA3, LFY:
LEAFY, MYB4: MYB DOMAIN PROTEIN 4, PI: PISTILLATA, PIF4: PHYTOCHROME
INTERACTING FACTOR 4, PIF5: PHYTOCHROME INTERACTING FACTOR 5, PRR5:
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PSEUDO-RESPONSE REGULATOR 5, SEP3: SEPALLATA3, SR1/CAMTA3: SIGNAL
RESPONSIVE 1/CALMODULIN-BINDING TRANSCRIPTION ACTIVATOR 3, TOC1:
TIMING OF CAB EXPRESSION 1.
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