DNA replication and chromosome positioning …...2020/04/02 · 4 103 and Pombo, 2019). It should be noted, however, that Hi-C identifies genome loci that are 104 associated in 3D

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DNA replication and chromosome positioning throughout the interphase in three-1

dimensional space of plant nuclei 2

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Němečková Alžběta, Veronika Koláčková, Vrána Jan, Doležel Jaroslav, Hřibová Eva* 5

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Institute of Experimental Botany of the Czech Academy of Sciences, Centre of the Region 8

Hana for Biotechnological and Agricultural Research, Olomouc, Czech Republic 9

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Short Title: DNA replication and interphase chromosome positioning 11

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*Corresponding Author: 13

Eva Hřibová 14

Institute of Experimental Botany 15

Šlechtitelů 31 16

779 00 Olomouc 17

Czech Republic 18

Tel: +420 585 238 713 19

E-mail:[email protected] 20

21

E-mail addresses: 22

Němečková A. - [email protected] 23

Koláčková V. - [email protected] 24

Vrána J. - [email protected] 25

Doležel J. - [email protected] 26

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28

Highlight 29

Telomere and centromere replication timing and interphase chromosome positioning in seven 30

grass species differing in genome size indicates a more complex relation between genome size 31

and the chromosome positioning. 32

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.CC-BY-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted April 2, 2020. . https://doi.org/10.1101/2020.04.02.021857doi: bioRxiv preprint

https://doi.org/10.1101/2020.04.02.021857

http://creativecommons.org/licenses/by-nd/4.0/

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Abstract 35

Despite the recent progress, our understanding of the principles of plant genome organization 36

and its dynamics in three-dimensional space of interphase nuclei remains limited. In this 37

study, DNA replication timing and interphase chromosome positioning was analyzed in seven 38

Poaceae species differing in genome size. A multidisciplinary approach combining newly 39

replicated DNA labelling by EdU, nuclei sorting by flow cytometry, three-dimensional 40

immuno-FISH, and confocal microscopy revealed similar replication timing order for 41

telomeres and centromeres as well as for euchromatin and heterochromatin in all seven 42

species. The Rabl configuration of chromosomes that lay parallel to each other and their 43

centromeres and telomeres are localized at opposite nuclear poles, was observed in wheat, oat, 44

rye and barley with large genomes, as well as in Brachypodium with a small genome. On the 45

other hand, chromosomes of rice with a small genome and maize with relatively large genome 46

did not assume proper Rabl configuration. In all species, the interphase chromosome 47

positioning inferred from the location of centromeres and telomeres was stable throughout the 48

interphase. These observations extend earlier studies indicating a more complex relation 49

between genome size and interphase chromosome positioning, which is controlled by factors 50

currently not known. 51

52

53

Introduction 54

One of the exciting features of eukaryotic genomes is their organization in three-55

dimensional space of cell nuclei and changes during cell cycle. The chromatin organization 56

and positioning of individual chromosomes in the nucleus was a great enigma until recently. 57

Nevertheless, based on microscopic observations of dividing cells of Salamandra maculata 58

and Proteus anguinus, Carl Rabl predicted already in 1885 that the positioning of 59

chromosomes in interphase nuclei follows their orientation in the preceding mitosis 60

(Reviewed by Cremer et al., 2006). The hypothesis was confirmed later by cytogenetic 61

studies in human, animals as well as in plants with larger chromosomes, which demonstrated 62

that centromeric and telomeric regions cluster at opposite poles of interphase nuclei (Cremer 63

et al., 1982; Schwarzacher and Heslop-Harrison 1991; Werner et al., 1992). This arrangement 64

of chromosomes in interphase was dubbed the Rabl configuration. 65

Interestingly, a different arrangement of chromosomes in interphase nuclei was 66

observed in Arabidopsis thaliana, a model plant with small genome (1C~157 Mbp). Here, the 67

centromeres are located at the nuclear periphery, whereas the telomeres congregate around 68


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nucleolus (Armstrong et al., 2001; Fransz et al., 2002). Centromeric heterochromatin forms 69

dense bodies called chromocenters, while euchromatin domains form 0.2 - 2 Mb loops that 70

are organized into Rosette-like structures (Fransz et al., 2002; Pecinka et al., 2004; Tiang et 71

al., 2012; Schubert et al., 2014). 72

Some earlier studies suggested that chromatin arrangement in interphase nuclei of 73

wheat, oat and rice, and in particular the centromere - telomere orientation, may be tissue-74

specific and cell cycle-dependent (Dong and Jiang 1998; Prieto et al., 2004; Santos and Show, 75

2004). While a majority of nuclei in somatic cells of rice (Oryza sativa), which has a small 76

genome (1C~490 Mbp, Bennett et al., 1976) lack Rabl configuration, chromosomes in the 77

nuclei of some rice tissues, e.g., pre-meiotic cells in anthers or xylem - vessel precursor cells 78

seem to assume the configuration (Prieto et al., 2004; Santos and Show 2004). Such 79

chromosome arrangement and orientation in somatic cell nuclei was also observed in other 80

plants with small genomes, including Brachypodium distachyon (1C~ 355 Mbp) (Idziak et al., 81

2015). Although the majority of cell types of small plant genomes of B. stacei (1C~276 Mbp, 82

Catalán et al., 2012) and B. hybridum (1C~619 Mbp, Catalán et al., 2012) did not show Rabl 83

configuration. On the other hand, recent study of Idziak et al. (2015) showed that Rabl 84

configuration is present in small proportion (13 and 17%) of root meristematic cells of both 85

these plant species. Rabl configuration was not observed also in plants with large genomes 86

such as Allium cepa (~ 1.5 Gb), Vicia faba (~ 15Gb), Solanum tuberosum (~ 900 Mbp) and 87

Pisum sativum (~ 3.9 - 4.4 Gb) (Rawlins et al., 1991; Fussell, 1992; Kamm et al., 1995; 88

Harrison and Heslop-Harrison, 1995). 89

Development of a three-dimensional fluorescence in situ hybridization (3D-FISH) 90

method provided an opportunity to map telomere positions at meiotic prophase in maize (Bass 91

et al., 1997), Arabidopsis and oat (Howe et al., 2013). In order to characterize changes in 92

chromosome positioning in 3D nuclear space throughout the interphase, cell nuclei at 93

particular cell cycle phase can be isolated using flow cytometry. Embedding flow-sorted 94

nuclei in polyacrylamide gel stabilizes their structure during the 3D-FISH procedure and 3D 95

images can be captured by confocal microscopy (Kotogány et al., 2010; Hayashi et al., 2013; 96

Bass et al., 2014; Koláčková et al., 2019). 97

Chromosome conformation capture (3C) technique (Dekker 2002) and its variants 98

offer an alternative approach to study spatial organization of chromatin in cell nuclei. So 99

called Hi-C method (Lieberman-Aiden et al., 2009) analyses contacts between DNA loci 100

across the whole genome. The contact maps thus obtained enable the analysis of chromosome 101

contact patterns, genome packing and 3D chromatin architecture (Dong et al., 2018; Kempfer 102


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and Pombo, 2019). It should be noted, however, that Hi-C identifies genome loci that are 103

associated in 3D space, but it does not provide the information on their physical position in 104

the nuclei. 105

In a majority of cases, spatial organization of chromatin in interphase nuclei revealed 106

by FISH corresponded to the results obtained by Hi-C (Sexton et al., 2012; Dong et al., 2017; 107

Mascher et al., 2017; Liu et al., 2017). The only exception was Arabidopsis thaliana where 108

Hi-C analysis did not confirm the Rosette-like organization of chromosomal domains. 109

According to Hi-C, telomeres of different chromosomes should cluster, but FISH studies 110

show telomeres located around nucleolus (Feng et al., 2014; Liu and Weigel et al., 2015). 111

Several investigations focused on organization of chromatin, its structure and changes 112

during cell cycle. In mammals, DNA sequences located at interior regions of nuclei are 113

replicated earlier, while late replication occurred mostly in nuclear periphery (Gilbert et al., 114

2010; Bryant and Aves 2011). Cell cycle kinetics and the progression of cells through S phase 115

can be followed in detail after labelling newly synthesized DNA by a thymidine analogue 5-116

ethynyl-2´-deoxyuridine (EdU) and subsequent bivariate flow cytometric analysis of nuclear 117

DNA content and the amount of incorporated EdU (Mickelson-Young et al., 2016). EdU-118

labelled nuclei can be sorted by flow cytometry and used as templates for FISH to analyze 119

replication timing of particular DNA sequences and their positioning in 3D nuclear space 120

(Hayashi et al., 2013; Bass et al., 2014; Bass et al., 2015; Dvořáčková et al., 2018). 121

Using 3D microscopy to analyze DNA replication dynamics in maize root meristem 122

cells, Bass et al. (2015) observed distinct patterns of EdU signal distribution during early and 123

middle S phase. In early S phase, DNA replication primarily occurred in regions characterized 124

by weak DAPI fluorescence, while replication in middle S phase correlate with strong DAPI 125

signals. Authors also revealed that knob regions and centromeric regions associated with 126

heterochromatin were replicated during late S phase. Based on their findings they proposed 127

“mini-domain model”, when gene islands are replicated in early S phase, and blocks of 128

repetitive DNA in middle S phase (Bass et al., 2015). 129

DNA replication dynamics was analyzed in detail by microscopy in several plant 130

species, including monocot species maize and barley, and dicots Arabidopsis and field bean 131

(Jasencakova et al., 2001; Bass et al., 2014; Jacob et al., 2014; Robledillo et al., 2018). In 132

general, different stages of S phase contrasted in DNA replication pattern. The early S phase 133

was characterized by weak dispersed signals of EdU, speckled signals of EdU were typical for 134

late S phase, while the nuclei in the middle S phase were all covered with EdU signals, except 135


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of nucleolar area (Jasencakova et al., 2001; Kotogány et al., 2010; Bass et al., 2014; Bass et 136

al., 2015;). 137

Interestingly, the dynamics of interphase chromosome positioning during cell cycle in 138

plants was not studied to date. In order to fill this gap, we characterized chromosome 139

positioning in 3D nuclear space during cell cycle and identified replication timing of DNA in 140

centromeric and telomeric regions. To do this, we labelled newly synthesized DNA by EdU, 141

followed cell cycle kinetics by flow cytometry and performed 3D immuno-FISH on flow-142

sorted nuclei. Chromosome positioning was analyzed in interphase nuclei of root meristem 143

cells in seven Poaceae species differing in nuclear genome size. Confocal microscopy of 144

nuclei embedded in polyacrylamide gel allowed us to locate centromeres and telomeres in 3D 145

space and to analyze replication timing of these chromosome regions. The results provided 146

new information on chromosome positioning and spatio-temporal pattern of DNA replication 147

of the important chromosome domains. 148

149

Keywords 150

DNA replication, EdU labelling, flow cytometry, Poaceae, Rabl configuration, S phase, three-151

dimensional fluorescence in situ hybridization (3D-FISH) 152

153

Abbreviations 154

3D – three-dimensional 155

CenH3 – centromere-specific variant of histone H3 156

BrdU – 5-bromo-2'-deoxyuridine 157

EdU – 5-ethynyl-2'-deoxyuridine 158

FISH – fluorescence in situ hybridization 159

PAA – polyacrylamide 160

rDNA – ribosomal deoxyribonucleic acid 161

ROI – region of interest 162

RT – room temperature 163

164

Material and methods 165

Plant material and seeds germination 166

Plants used in the present study included wheat (Triticum aestivum L.) cultivar Chinese 167

Spring (2n=2x=42), oat (Avena sativa L.) cultivar Atego (2x=2x=42), barley (Hordeum 168


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vulgare L.) cultivar Morex (2n=2x=14), rye (Secale cereale L.) cultivar Dánkowskie Diament 169

(2n=2x=14), rice (Oryza sativa L.) cultivar Nipponbare (2n=2x=24), maize (Zea mays L.) line 170

B73 (2n=2x=20) and Brachypodium distachyon L. cultivar Bd21 (2n=2x=10). All seeds were 171

obtained from IPK Genebank (Gaterleben, Germany) except for rice, which was kindly 172

provided by prof. Takashi Ryu Endo (Kyoto University, Kyoto, Japan) and wheat, which was 173

obtained from Wheat Genetics & Genomic Resources Center (Kansas state university, 174

Manhattan, KS 66506). All seeds were germinated in a biological incubator at 24 °C in glass 175

Petri dishes on moistened filter paper until the primary roots were 2.5 – 4 cm long. 176

177

EdU labelling of replicating DNA 178

Young seedlings were incubated in 20 µM 5-ethynyl-2′-deoxyuridine (EdU) (Click-iT™ EdU 179

Alexa Fluor™ 488 Flow Cytometry Assay Kit, ThermoFisher Scientific/Invitrogen, Waltham, 180

Massachusetts, USA) made in ddH2O for 30 min at 24 °C. Root tips were excised and fixed in 181

2% (v/v) formaldehyde in Tris buffer for 20 min at 4°C, and washed 3 times in Tris buffer at 182

4°C (Doležel et al., 1992). About ten root tips with incorporated EdU were treated in 0.5 ml 183

Click-iT reaction cocktail (Click-iT™ EdU Alexa Fluor™ 488 Flow Cytometry Assay Kit) 184

prepared according to the manufacturer instructions, incubated for 10 min under vacuum, 185

followed by incubation in the dark for 45 min at room temperature (RT). After the labelling, 186

roots were washed 3 times for 5 min in phosphate buffered saline (PBS; 10 mM Na2HPO

4, 187

2mM KH2PO

4, 137mM NaCl, 2.7mM 188

KCl, pH 7.4) on a rotating shaker (160 rpm) at RT. 189

The root tips were mounted in 0.1 % agarose made in re-distilled water onto cavity 190

microscopic slides (Paul Marienfeld GmbH & Co. KG, Lauda-Königshofen, Germany) and 191

mounted in Vectashield with DAPI (Vector Laboratories, Ltd., Peterborough, UK) to 192

counterstain the chromosomes. The preparations were imaged using a Leica TCS SP8 STED 193

3X confocal microscope (Leica Microsystems, Wetzlar, Germany) with 10x/0.4 NA Plan-194

Apochromat objective (z-stacks, pinhole Airy). Image stacks were captured separately for 195

DAPI using 405 nm laser and for EdU labelled by Alexa Fluor 488 using 488 nm laser, and 196

appropriate emission filters. Typically, image stacks of 36 slides in average with 138 µm 197

spacing were acquired. Finally, maximum intensity projections were done using Leica LAS-X 198

software and final image processing was done using Adobe Photoshop 6 (Adobe Systems). 199

200

EdU labelling of replicating DNA and nuclei preparation for flow cytometry 201


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Roots of young seedlings were incubated in 20 µM EdU made in ddH2O (Click-iT™ EdU 202

Alexa Fluor™ 488 Flow Cytometry Assay Kit) for 30 min at 24 °C. Suspensions of intact 203

nuclei were prepared according to Doležel et al. (1992). Briefly, ~1 cm-long root tips were 204

cut and fixed with 2% (v/v) formaldehyde in Tris buffer (10 mM Tris, 10 mM Na2EDTA, 100 205

mM NaCl, 0.1% Triton X-100, 2 % formaldehyde, pH 7.5) at 4°C and washed three times 206

with Tris buffer at 4°C. Meristematic parts of root tips (~1 mm-long) were excised from 70 207

roots per sample in barley, oats, wheat, rye, and from 100 roots in rice and Brachypodium. 208

Root meristems were homogenized in 500 µl LB01 buffer (Doležel et al., 1989) by Polytron 209

PT 1200 homogenizer (Kinematica AG, Littua, Switzeland) for 13 s at 10 000 – 24 000 rpm 210

depending on a species. For maize, 50 root meristems were chopped using a razor blade 211

according to Doležel et al. (1989). The crude homogenates were passed through 50 µm nylon 212

mesh and nuclei were pelleted at 500 g, at 4 °C for 10 min. The pellet was resuspended in 0.5 213

ml Click-iT reaction cocktail prepared according to the manufacturer instructions and the 214

nuclei were incubated in the dark at 24 °C for 30 min. Then the nuclei were pelleted at 500 g 215

for 10 min, resuspended in 500 µl of LB01 buffer and stained by DAPI (0.2 µg/ ml final 216

concentration). Finally, the suspensions were filtered through a 20 µm nylon mesh and 217

analyzed using FASCAria II SORP flow cytometer and sorter (BD Bioscience, San Jose, 218

USA) equipped with a UV (355 nm) and blue (488 nm) lasers. Nuclei representing different 219

phases of cell cycle were sorted into 1x meiocyte buffer A (1x buffer A salts, 0.32M sorbitol, 220

1x DTT, 1x polyamines) (Bass et al., 1997; Howe et al., 2013). 221

222

Nuclei mounting in polyacrylamide gel 223

Flow sorted nuclei were mounted in 5% polyacrylamide (PAA) gel as described by Howe et 224

al. (2013) and Bass et al. (1997) with minor modifications. Briefly, 500 µl PAA mix 225

containing 15 % (w/v) acrylamide/bisacrylamide (akrylamid/bisakrylamid 30% NF 226

ROTIPHORESE (29 : 1) Roche Applied Science, Penzberg, Germany), 1x Buffer A salts (10 227

x buffer contains 800 mM KCl, 200 mM NaCl, 150 mM PIPES, 20 mM EGTA, 5 mM 228

EDTA, 1 M NaOH, pH 6.8), 1x polyamines (1,000 x polyamines contains 0.15 M spermine 229

and 0.5 M spermidine), 1x dithiothreitol (1,000 x dithiothreitol contains 1.0 M DTT, 0.01 M 230

NaOAc), 0.32 M sorbitol and 99 µl ddH2O was rapidly combined with 25 µl of freshly 231

prepared 20% ammonium sulfate in ddH2O and 25 µl 20% sodium sulfate (anhydrous) in 232

ddH2O. One volume of activated PAA gel mix was mixed with two volumes of flow-sorted 233

nuclei on a microscopic slide coated with aminoalkylsilane (Sigma-Aldrich, Darmstadt, 234


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Germany) and stirred gently with the pipette tip. The PAA drop was covered with a clean 235

glass coverslip and let to polymerize at 37°C for ~40 min. The coverslip was removed and 236

silane-coated slides with acrylamide pad were washed three times with 1x MBA in Coplin jar 237

to remove unpolymerized acrylamide. 238

239

Immuno-staining and fluorescence in situ hybridization (FISH) 240

In order to visualize centromeric regions, slides with PAA pad were washed in blocking 241

buffer (phosphate buffer, 1% Triton X-100, 1 mM EDTA) at RT for 1 h, after which 100 µl of 242

blocking buffer was added per slide and covered with parafilm for 10 min. Next, 50 µl of 243

diluted anti-OsCenH3 primary antibody (1:100) (Nagaki et al., 2004) was added, covered 244

with parafilm and incubated at 4°C in a humid chamber for 12 hrs. The slides were then 245

washed in a wash buffer (phosphate buffer, 0.1% Tween, 1 mM EDTA) three times for 15 246

min and once for 10 min in 2x Saline-Sodium Citrate buffer (2x SSC) and fixed in 1% (v/v) 247

formaldehyde in 2 x SSC for 30 min at RT. After the fixation, slides were washed 3 x 15 min 248

in 2x SSC at RT and used for FISH. 249

Hybridization mix (35 µl) containing 50% formamide, 2x SSC and 400 ng of directly 250

labelled telomere oligo-probe ([CCCTAAA]4) was added onto a slide and covered by a glass 251

coverslip. The slides were denatured at 94 °C for 6 min and incubated in a humid chamber at 252

37 °C overnight. Post-hybridization washing steps comprised of 5 min wash in 2x SSC, 253

stringent washes 2 x 15 min in 0.1 x SSC at 37°C, 2 x 15 min in 2x SSC at 37 °C, 2 x 15 min 254

2x SSC at RT, and 1 x 15 min in 4x SSC at RT. After washing, 100 µl of secondary antibody 255

diluted 1:250 in blocking buffer (5 % bovine serum albumin with 0.1 % Tween dissolved in 256

phosphate buffer, secondary antibody anti-Rabbit Alexa Fluor 546 (ThermoFisher 257

Scientific/Invitrogen) was added, covered with parafilm and incubated at 37 °C for 3h. 258

Finally, the preparation was washed in 4x SSC (3 x 15 min, RT) and in phosphate buffer (3 x 259

15 min, RT). PAA pad was mounted in 30 µl of Vectashield with DAPI (Vector 260

Laboratories), covered with a glass coverslip, and sealed with nail polish. 261

262

Confocal microscopy and image analysis 263

Images were acquired using Leica TCS SP8 STED 3X confocal microscope (Leica 264

Microsystems) equipped with 63x/1.4 NA Oil Plan-Apochromat objective (z-stacks, pinhole 265

Airy) equipped by Leica LAS-X software with Leica Lightning module. Image stacks were 266

captured separately for each fluorochrome using 647 nm, 561 nm, 488 nm, and 405 nm laser 267

lines for excitation and appropriate emission filters. Typically, image stacks of 100 slides on 268


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average with 0.2 µm spacing were acquired. 3D models of microscopic images, colocalization 269

analysis and volume calculations were performed using Imaris 9.2 software (Bitplane, Oxford 270

Instruments, Zurich, Switzerland). To estimate colocalization signals, Imaris software’s 271

‘Colocalization’ function based on Pearson’s correlation coefficient were used (Manders et 272

al., 1992). The region of interest (ROI) was individually determined for each nucleus and 273

each channel. Importantly, setting ROI ensures, the ‘layer by layer’ correlation, so negative 274

colocalization of green channel of EdU signals representing another layer is prevented. The 275

volume of each nucleus and EdU signals were detected based on primary intensity of 276

fluorophores obtained after microscopic analysis. Imaris function ‘Surface’ and ‘Spot 277

detection’ was used for modeling the centromere - telomere arrangements. Chanel contrast 278

was adjusted using ‘Chanel Adjustment’ and videos were created using ‘Animation function’. 279

Between 100 and 150 nuclei were analyzed per each plant species. 280

281

282

Results 283

As we have studied plant species differing in genome sizes and root morphology, 284

experimental protocols had to be individually optimized. While EdU concentration and 285

incubation times were the same for all species, microscopic analysis of root tips after EdU 286

labelling revealed differences in the area of meristematic zones (Fig. 1). This information was 287

useful for the excision of meristematic regions to prepare suspensions of meristem cell nuclei. 288

Other critical step of the preparation of nuclei suspensions was the extent of mechanical 289

homogenization, which affected the nuclei integrity and yield (Table 1). The thick maize roots 290

were chopped in nuclei isolation buffer using a razor blade to obtain sufficient amounts of 291

nuclei suitable for flow cytometry. 292

DNA replication kinetics in interphase nuclei of root tip meristems during cell cycle 293

was evaluated after EdU incorporation into replicating DNA. Bivariate flow cytometric 294

analysis EdU vs. DAPI fluorescence resulted in typical “horseshoe” dotplot patterns, which 295

made it possible to unambiguously distinguish the nuclei at G1 and G2 phases of the cell 296

cycle and in early, middle and late S phase (Fig. 2). Fluorescent detection of incorporated 297

EdU was useful not only for flow cytometric analysis and nuclei sorting, but also for 298

microscopy. 299

300

DNA replication kinetics 301


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Microscopic analysis of EdU fluorescence in cell nuclei revealed different replication 302

patterns specific for early, middle and late S phase. Weak discrete signals were typical for 303

early DNA replication stage, while speckled signals concentrated in particular areas were 304

characteristic for late DNA replication stages. Strong signals dispersed throughout the whole 305

nuclei were observed in nuclei at middle S phase (Fig. 3). Overlaps between heterochromatin 306

regions and EdU signals at late S phase indicated that these regions were replicated later than 307

euchromatin. EdU signals were also detected inside nucleoli as discrete and well visible spots 308

during early and middle S phase. Clear signals were also seen at the periphery of nucleoli at 309

middle and late S phase (Supplementary Fig. 1), suggesting the replication of 45S rDNA loci. 310

311

Replication timing of centromeric and telomeric regions 312

Replication timing of centromeric and telomeric regions was determined after 313

microcopy of nuclei at different stages of S phase and based on the overlap of EdU and 314

immunofluorescent signals at centromeric regions and FISH signals at telomeric regions (Fig. 315

4). Although the replication of centromeric regions initiated at early S phase, the highest 316

number of centromeric regions underwent replication during middle and late S phase (Fig. 4, 317

5, Supplementary Fig. 2, 3, 4). In contrast, prevalent replication of telomeric sequences was 318

observed during early and middle S phase. This replication pattern of both chromosome 319

domains was observed in all species (Fig. 4, 5, Supplementary Fig. 5, 4, 7) except of rye, 320

where a minor difference was observed in replication dynamics of telomeric sequences. 321

Telomeric regions of rye comprise large heterochromatic blocks (Appels et al., 1978; 322

Evtushenko et al., 2016) and our results showed, that DNA loci closely connected to 323

heterochromatic block (probably flanking regions) were replicated at late S phase, while 324

telomeres located out of heterochromatin were replicated earlier, during middle S phase (Fig. 325

4B, D, E). 326

The difference in phasing centromere and telomere replication had an impact on the 327

overall replication pattern of S phase nuclei as highlighted by EdU (Supplementary videos 1, 328

2, 3). The first replication signals in the nuclei were concentrated at telomeric regions, while 329

the opposite pole of nucleus where centromeres were localized lacked EdU signals. In 330

analogy, heterochromatin blocks and regions around centromeres replicated during late S 331

phase, whereas the telomeres at the opposite pole of nuclei lacked the replication signals. A 332

majority of nuclear DNA replicated during middle S phase, resulting in strong EdU signals 333

spread across the whole nuclear volume (Supplementary Video 4, 5, 6). The images captured 334

in rye are shown in Figure 4; supplementary Figures 2 – 7 show the images captured in the 335


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remaining species. The analysis of FISH signals by the Imaris software (Fig. 5) showed that 336

co-localization between EdU and centromere fluorescence channels increased linearly from 337

early S phase to middle and late S phase. 338

339

Chromosome positioning in interphase nuclei 340

The localization of centromeres and telomeres during the course of S phase was used 341

to infer chromosome positioning in interphase nuclei. In total, we analyzed 700 nuclei (100 342

nuclei for each species) with spherical shapes which are typical for the meristem cells of the 343

root meristems. We confirmed regular Rabl configuration, when centromeres and telomeres 344

localize at opposite nuclear poles, in large genomes of wheat, oat, rye, barley, as well as in 345

Brachypodium distachyon with a small genome. On the other hand, chromosomes of rice with 346

a small genome and maize with relatively large genome did not assume proper Rabl 347

configuration (Fig. 6). The given interphase chromosome arrangement was stable throughout 348

the interphase in all species (Fig. 7, Supplementary Fig. 8, Supplementary Video 1 – 12). 349

In a majority of species, the number of fluorescence signals from centromeres and 350

telomeres corresponded to the number of mitotic chromosomes. The only exception was rice, 351

where the Rabl configuration was not observed. Here, the telomeric and centromeric signals 352

constituted large clustered signals randomly distributed in the nucleoplasm (Fig. 6, 353

Supplementary Video 7, 8, 9). In maize, the centromeres clustered in one region of nuclei, but 354

telomeres were randomly dispersed over the whole nucleoplasm (Fig. 6, Supplementary 355

Video 10, 11, 12). 356

357

Discussion 358

There is a growing interest to understand the principles of genome organization and its 359

dynamics in three-dimensional space of interphase nuclei at various levels: from DNA fibers 360

up to individual chromosomes and their domains. A range of studies focused on interphase 361

chromosome positioning in plants (e.g. Pecinka et al., 2004; Idziak et al., 2015). However, 362

except a study on maize (Bass et al., 2015), chromosome positioning was not followed 363

throughout the interphase, from G1 to G2 phase of the cell cycle. In order to fill these gaps, 364

we analyzed spatiotemporal patterns of DNA replication and positioning of interphase 365

chromosomes during cell cycle in root tip meristems of seven Poaceae species. They included 366

important and evolutionary related crops differing in genome size and Brachypodium 367

distachyon, a model wild species with a small genome. 368


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A popular approach to study patterns of DNA replication in different parts of cell 369

nuclei was microscopic detection of thymidine analogues incorporated into the newly 370

synthesized DNA (Gilbert et al., 2010; Bryant and Aves 2011; Bass et al., 2014; Bass et al., 371

2015). In mammals, early replication was observed in the interior regions of nuclei, while late 372

replication occurred mostly at nuclear periphery (Li et al., 2001; Pope and Gilbert 2013). In 373

plants, differences in replication patterns at early, middle and late S phase were revealed by 374

fluorescently labelled thymidine analogue 5-ethynyl-2'-deoxyuridine (EdU) (Hayashi et al., 375

2013; Bass et al., 2014; Bass et al., 2015; Dvořáčková et al., 2018). However, with a few 376

exceptions, the patterns of nuclear DNA replication were analyzed only in plants with small 377

and moderate genome sizes, such as Arabidopsis, rice and maize (Hayashi et al., 2013; Bass 378

et al., 2014; Bass et al., 2015; Dvořáčková et al., 2018). 379

Earlier, Cortes et al. (1980) used thymidine analogue 5-bromo-2'-deoxyuridine (BrdU) 380

in Allium cepa to observe a high coincidence between constitutive heterochromatin, including 381

pericentromeric regions and late replicating DNA, which possesses a large genome. In barley, 382

another species with a large genome, Jasencakova et al. (2001) found that DNA replication 383

started at rDNA loci, continued at euchromatin and centromeric regions and was completed at 384

pericentromeric heterochromatin. Our observations obtained after EdU labelling of newly 385

replicated DNA agree with the results obtained in maize by Bass et al. (2015). Early S phase 386

nuclei were characterized by localized weak signals, strong signals dispersed throughout the 387

whole nuclei were observed in the nuclei at middle S phase and speckled signals concentrated 388

in particular areas were observed at late DNA replication stages. As we studied seven species 389

differing considerably in genome size, our results indicate that this pattern of DNA replication 390

is general and does not depend on the amount of nuclear DNA. 391

We combined EdU labelling with the localization of telomere and centromere regions 392

by FISH to provide a more detailed view on DNA replication kinetics. We observed opposite 393

replication timing of telomeres and centromeres in all seven plant species, where the highest 394

intensity of early replication was observed in gene-dense chromosome termini, while the 395

highest intensity of late replicating DNA was typical for pericentromeric regions. In middle S 396

phase, replication was almost evenly dispersed along the entire chromosomes and only 397

slightly increased in the interstitial regions of chromosome arms. These findings confirmed 398

the recent results made in Arabidopsis and maize obtained by Repli-seq analysis and 399

corresponded to the gene density of their chromosome profiles (Wear et al., 2017, Zynda et 400

al., 2017; Concia et al., 2018). Kwasniewska et al. (2018) showed that terminal parts of 401

barley chromosomes replicated in early S phase, whole chromosomes were covered with EdU 402


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13

signal at middle S phase and centromeric parts of chromosomes were replicated in late S 403

phase. The authors reasoned that this chromosome replication profile implied presence of 404

transcriptionally active genes in the terminal parts of chromosomes and inactive 405

heterochromatin in the centromeric regions. 406

Two main types of arrangement were described for chromosomes in interphase nuclei 407

of plants: Rabl configuration and Rosette-like structure (Rabl 1885; Francz et al., 2002; Tiang 408

et al., 2012). The Rabl configuration, where centromeres and telomeres localize at opposite 409

nuclear poles is considered typical for plants with large genomes. However, this does not 410

seem to be a general rule and in some plants with relatively large genomes such maize and 411

sorghum, Rabl configuration was not confirmed (Anamthawat-Jónsson and Heslop-Harrison 412

1990; Schubert and Shaw 2011; Tiang et al., 2012). The Rosette structure, where the 413

centromeres are located at the nuclear periphery whereas the telomeres congregate around 414

nucleolus was described only in Arabidopsis (Armstrong et al., 2001; Fransz et al., 2002). 415

This work revealed a stable arrangement of centromeres and telomeres throughout the 416

interphase. Based on the positions of telomeres and centromeres, we confirmed Rabl 417

configuration in Brachypodium distachyon with a small genome, and in barley, rye, oat and 418

wheat with large genomes. We also confirmed, that chromosomes in rice with a small genome 419

and maize with a moderate genome did not assume Rabl configuration, confirming the results 420

of Dong and Jiang (1998) and Santos and Shaw (2004) obtained by FISH on interphase 421

nuclei. Some of the centromeric signals clustered at specific region of nucleoplasm, indicating 422

a tendency to Rabl-like polarized organization, but telomeric signals were dispersed. One 423

reason for the irregular distribution of rice and maize interphase chromosomes could be the 424

presence of acrocentric chromosomes as hypothesized by Idziak et al., (2015) who observed 425

disrupted Rabl configuration in Brachypodium stacei and B. hybridum whose karyotypes 426

comprise acrocentric chromosomes. 427

To conclude, our study indicates that spatiotemporal pattern of DNA replication 428

timing during S phase in plants is conserved and does not depend on the amount of nuclear 429

DNA. While the positioning of interphase chromosomes is stable throughout cell cycle, there 430

seems to be more complex relation between interphase chromosome positioning and genome 431

size. The observations by other authors that chromosome positioning may differ between 432

tissues and even within tissue of the same plant indicates that interphase chromosome 433

configuration is not a simple consequence of chromosome orientation in the preceding mitosis 434

and that it is controlled by so far unknown factors. 435

436


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14

437

Acknowledgements 438

We are grateful to Dr. Kateřina Malínská for advice on confocal microscopy and we thank 439

Ms. Zdeňka Dubská and Bc. Jitka Weiserová for excellent technical assistance. We 440

acknowledge the core facility CELLIM of CEITEC, which has been supported by the Czech-441

BioImaging large RI project funded by MEYS CR, grant award LM2015062 for providing 442

imaging facility. This work was supported by the Czech Science Foundation (grant award 17 -443

14048S) and by the ERDF project "Plants as a tool for sustainable global development" (grant 444

award CZ.02.1.01/0.0/0.0/16_019/0000827). 445

446

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597

Legends to figures 598

Table 1 599

Parameters for nuclei suspension preparation after EdU pulse for individual species. 600

601

Figure 1 602


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20

Root meristematic zones of seven Poaceae species. Roots were incubated with 20 mM EdU 603

for 30 min and EdU incorporated into replicating DNA was detected by Alexa Fluor 488 604

(green color). Nuclei were stained with DAPI (blue color). 605

606

Figure 2 607

Bivariate flow cytometric analysis of cell cycle in rye (A) and rice (B). Roots of young 608

seedlings were incubated with 20 mM EdU for 30 min. EdU in isolated nuclei was detected 609

by Alexa Fluor 488 (green color) and their DNA was stained with DAPI (blue). x axis 610

represents relative DNA content estimated as the intensity of DAPI fluorescence (linear 611

scale). y axis shows the extent of EdU incorporation into newly synthesized DNA quantified 612

by Alexa Fluor 488 fluorescence intensity (log scale). Red boxes in the dot plot show G1- and 613

G2-phase nuclei, green boxes highlight the early, middle and late S phase. 614

615

Figure 3 616

Maximum intensity projection of rye nuclei in 3D in different phases of cell cycle. EdU 617

(green color) was incorporated during a 30 min pulse into the newly synthesized DNA. G1 618

and G2 phases lack green signals of EdU. Changes in DNA replication pattern during early, 619

middle and late S phase are clearly visible. Note the replication of heterochromatin regions. 620

DNA was stained by DAPI (blue color). 621

622

Figure 4 623

DNA replication in rye nuclei and replication timing of centromeric and telomeric sequences. 624

Colocalization of signals specific to telomeres (red) and centromeres (pink) with EdU signals 625

corresponding to replicated DNA (green) was used to describe the pattern of replication 626

timing. In early S phase, centromeric signals (A) as well as telomeric signals which are 627

connected with heterochromatin regions (B, red rectangle) did not colocalize with EdU. 628

Telomeric sequences which were not localized in heterochromatin (B, white rectangle) co-629

localized with EdU signals, pointing to ongoing replication process. Colocalization of EdU 630

and cetromeric signals is visible in middle and late S phase (C, D). Similarly, telomeric 631

sequences connected with heterochromatin regions (red rectangle) colocalized with EdU in 632

mid and late S-phase (E, G). 633

634

Figure 5 635


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21

Replication timing of centromeric and telomeric sequences. Replication time was obtained 636

after colocalization analysis and volume calculations of 3D models of microscopic images by 637

Imaris 9.2 software. The columns represent percent of colocalized volume of signals from 638

telomeric and centromeric regions and EdU signals. 639

640

Figure 6 641

Chromosome positioning in G1 nuclei estimated based on the positions of telomeres and 642

centromeres. Centromeres were labelled using CenH3 antibody (yellow), telomeres were 643

visualized by FISH with an oligonucleotide probe (red color). Nuclear DNA was stained with 644

DAPI (blue color). 645

646

Figure 7 647

Orientation of chromosomes in interphase nuclei of rye. Centromeres were labelled using 648

CenH3 immunostaining (yellow), telomeres were visualized by FISH with oligonucleotide 649

probe (red color). Nuclear DNA was stained with DAPI (blue). 650

651

652

Supplementary data 653

654

Supplementary Figure S1 655

Replication of 45S rDNA in oat. EdU signals (green) and 45S rDNA FISH signals (red) 656

detected inside the nucleoli (white dashed lines) at early and middle S phase and in the 657

periphery of nucleoli at middle and late S phase. 658

659


Non-colocalization of CenH3 in early S phase in seven species. Immunofluorescent detection 661

of CenH3 (pink) and EdU signals corresponding to replicating DNA (green) were visualized 662

in Brachypodium distachyon (A), Oryza sativa (B), Zea mays (C), Hordeum vulgare (D), 663

Secale cereale (E), Avena sativa (F) and Triticum aestivum (G). Non-colocalized signals are 664

shown in white rectangles. 665

666


Colocalization of CenH3 in middle S phase in seven species. Immunofluorescent detection of 668

CenH3 (pink) and EdU signals corresponding to replicating DNA (green) were visualized in 669


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Brachypodium distachyon (A), Oryza sativa (B), Zea mays (C), Hordeum vulgare (D), Secale 670

cereale (E), Avena sativa (F) and Triticum aestivum (G). colocalization is shown by white 671

rectangles. 672

673

Supplemetary Figure S4 674

Colocalization of CenH3 in late S phase in seven selected species. Immunofluorescent 675

detection of CenH3 (pink) and EdU signals corresponding to replicating DNA (green) were 676

visualized in Brachypodium distachyon (A), Oryza sativa (B), Zea mays (C), Hordeum 677

vulgare (D), Secale cereale (E), Avena sativa (F) and Triticum aestivum (G). colocalization is 678

shown by white rectangle, 679

680


Colocalization of telomeres in early S phase in seven selected species. FISH signals specific 682

to telomeres (red) and with EdU signals corresponding to replicating DNA (green) were 683



shown by white rectangle and is visible as yellow color in merged pictures. 686

687


Colocalization of telomeres in middle S phase in seven selected species. FISH signals specific 689

to telomeres (red) and with EdU signals corresponding to replicating DNA (green) were 690



shown by white rectangle and is visible as yellow color in merged pictures. 693

694


Non-colocalization of telomeres in late S phase in seven selected species. FISH signals 696

specific to telomeres (red) and with EdU signals corresponding to replicating DNA (green) 697

were visualized in Brachypodium distachyon (A), Oryza sativa (B), Zea mays (C), Hordeum 698


shown by white rectangle. 700

701



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Models of 3D chromosome positioning in interphase nuclei of Brachypodium distachyon (A), 703

Oryza sativa (B), Zea mays (C), Hordeum vulgare (D), Secale cereale (E), Avena sativa (F) 704

and Triticum aestivum (G). The volume of nuclei (grey) was modeled based on the primary 705

intensity of DAPI staining. Telomeres (red) were detected based on the intensity of 706

fluorescently labelled telomeres and centromeres (yellow) were detected based on the primary 707

intensity of CenH3. 708

709


Maximum intensity projection of 3D nuclei from different stages of interphase in 711

Brachypodium distachyon (A), Oryza sativa (B), Zea mays (C), Hordeum vulgare (D), Secale 712

cereale (E), Avena sativa (F) and Triticum aestivum (G). All nuclei were counterstand with 713

DAPI (blue) and early, middle and late phase shows replication pattern visualized by EdU 714

(green). 715

716

Supplementary Video 1 717

Barley nucleus in early S phase. Centromeres were visualized using immunolabelling CenH3 718

(yellow), telomeres were visualized after FISH with an oligonucleotide probe (red) and 719

replicating DNA was labelled with EdU (green). Nuclear DNA was stained with DAPI (blue). 720

721


Barley nucleus in middle S phase. Centromeres were visualized using immunolabelling 723

CenH3 (yellow), telomeres were visualized after FISH with an oligonucleotide probe (red) 724

and replicating DNA was labelled with EdU (green). Nuclear DNA was stained with DAPI 725

(blue). 726

727


Barley nucleus in late S phase. Centromeres were visualized using immunolabelling CenH3 729



732


Rye nucleus in early S phase. Centromeres were visualized using immunolabelling CenH3 734




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737


Rye nucleus in middle S phase. Centromeres were visualized using immunolabelling CenH3 739



742


Rye nucleus in late S phase. Centromeres were visualized using immunolabelling CenH3 744


replicating was labelled with EdU (green). Nuclear DNA was stained with DAPI (blue). 746

747


Rice nucleus in G1 phase. Centromeres were visualized using immunolabelling CenH3 749

(yellow) and telomeres were visualized after FISH with an oligonucleotide probe (red). 750

Nuclear DNA was stained with DAPI (blue). 751

752


Rice nucleus in middle S phase. Centromeres were visualized using immunolabelling CenH3 754



757


Rice nucleus in G2 phase. Centromeres were visualized using immunolabelling CenH3 759



762


Maize nucleus in G1 phase. Centromeres were visualized using immunolabelling CenH3 764



767


Maize nucleus in middle S phase. Centromeres were visualized using immunolabelling 769

CenH3 (yellow), telomeres were visualized after FISH with an oligonucleotide probe (red) 770


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and replicating DNA was labelled with EdU (green). Nuclear DNA was stained with DAPI 771

(blue). 772

773


Maize nucleus in G2 phase. Centromeres were visualized using immunolabelling CenH3 775



778


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Table 1: Parameters for the nuclei suspension preparation

Plant species Number of roots Homogenisation [rpm] Time [s] Brachypodium distachyon 100 10000 13 Oryza sativa 100 14500 13 Zea mays 50 Chopped by razor blade - Hordeum vulgare 70 14500 13 Secale cereale 70 14500 13 Avena sativa 70 24000 13 Triticum aestivum 70 20000 13


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DNA replication and chromosome positioning …...2020/04/02 · 4 103 and Pombo, 2019). It should be noted, however, that Hi-C identifies genome loci that are 104 associated in 3D