1 Short title: 6 Affiliations - Plant Physiology · 5 Toal, Siobhan M. Brady, Anne B. Britt*(2) 6...

1

Short title 1

SOG1 links DNA damage response to organ regeneration 2

Authors 3

Ross A Johnson(1) Phillip A Conklin(1) Michelle Tjahjadi Victor Missirian Ted 4

Toal Siobhan M Brady Anne B Britt(2) 5

Affiliations 6

Department of Plant Biology UC Davis 1 Shields Ave Davis CA 95616 530-752-7

0699 (1) these authors contributed equally (2) abbrittucdavisedu 8

Title 9

SUPPRESSOR OF GAMMA RESPONSE 1 links DNA damage response to organ 10

regeneration 11

One-sentence summary 12

SOG1 governs the programmed breakdown and reconstruction of the root stem cell niche 13

after acute DNA damage 14

Author contributions 15

PAC RAJ SB and ABB designed the experiments RAJ and PAC performed 16

experiments and produced figures ABB RAJ and PAC wrote the manuscript ABB 17

RAJ PAC MT and SB edited the manuscript MT developed novel sog1 lines TT wrote 18

the read-trimming script and VM processed the transcriptomics data 19

Funding sources 20

Funding was provided by a grant to ABB from the National Science Foundation Division 21

of Molecular Biosciences (award 1158443) and to PAC from the Elsie Taylor Stocking 22

Fellowship 23

24

Plant Physiology Preview Published on December 8 2017 as DOI101104pp1701274

Copyright 2017 by the American Society of Plant Biologists

wwwplantphysiolorgon May 26 2020 - Published by Downloaded from Copyright copy 2017 American Society of Plant Biologists All rights reserved

2

Abstract 25

In Arabidopsis DNA damage-induced programmed cell death is limited to the 26

meristematic stem cell niche and its early descendants The significance of this cell-type 27

specific programmed cell death is unclear Here we demonstrate in roots that it is the 28

programmed destruction of the mitotically-compromised stem cell niche that triggers its 29

regeneration enabling growth recovery In contrast to wild-type plants sog1 plants 30

which are defective in damage-induced programmed cell death maintain the cell 31

identities and stereotypical structure of the stem cell niche after irradiation but these cells 32

fail to undergo cell division terminating root growth We propose DNA damage-induced 33

programmed cell death is employed by plants as a developmental response contrasting 34

with its role as an anti-carcinogenic response in animals This role in plants may have 35

evolved to restore the growth of embryos after the accumulation of DNA damage in 36

seeds 37

Keywords Programmed cell death ionizing radiation double-strand breaks stem 38

cell niche 39

40


3

Introduction 41

DNA damage can cause cytostatic and cytotoxic effects and can potentially lead to 42

heritable mutations (Waterworth et al 2015) Double-strand DNA breaks (DSBs) are 43

particularly growth-disruptive leading to chromosome aberrations and mutations if 44

incorrectly repaired and cell death if mitosis occurs before the repair of broken 45

chromosomes (Hu et al 2016 Waterworth et al 2015) DSBs are also potent inducers of 46

checkpoint response where cell cycle progression is transiently inhibited to allow for 47

DNA repair before mitotic M-phase (Hu et al 2016) These arrest and repair processes 48

together with programmed cell death (Curtis and Hays 2007) and the early induction of 49

the endoreduplicative cell cycle (Adachi et al 2011) are collectively known as the DNA 50

damage response (DDR) which safeguards the genomic integrity of the organism as a 51

whole (Yoshiyama 2016) In plants DDR has a key role in the germination of seeds that 52

have accumulated DNA damage during aging from desiccationrehydration cycles as 53

repair is limited in the desiccated state (Waterworth et al 2016 Waterworth et al 2015) 54

Here we demonstrate the role of DDR genes in seedling recovery from growth-disruptive 55

levels of DNA damage which we have artificially induced by exposure to ionizing 56

radiation (IR) In this work we have used gamma irradiation for a ubiquitous induction of 57

DNA damage throughout the seedling with DSBs being the most cytotoxic lesion 58

triggered (Tounekti et al 2001 Moiseenko et al 2001) We have typically used an 59

acute transiently growth-inhibiting 150 Gy dose which is less than that triggering a 60

permanent growth arrest (eg 500 Gy) but greater than that resolvable by constitutively 61

expressed DNA repair processes (eg 5 Gy) (Einset and Collins 2015) eukaryotic 62


4

genomes routinely encounter a benign level of DSBs (such as from collapsed DNA 63

replication forks) that do not induce the DDR (Sanchez et al 1999) 64

65

A plant cellrsquos response to DNA damage first involves the DSB-detecting protein kinase 66

ATM (Ataxia-Telangiectasia-Mutated) or the detector of stalled replication forks ATR 67

(Ataxia Telangiectasia Mutated and Rad3-related protein) (Culligan et al 2006 68

Furukawa et al 2010) ATM (Yoshiyama et al 2013) and inferably ATR (Furukawa et 69

al 2010 Yoshiyama et al 2009) can phospho-activate the transcription factor SOG1 as 70

well as other proteins in plant cells (Roitinger et al 2015 Yoshiyama et al 2013) Once 71

activated SOG1 transcriptionally-induces various functional classes of DDR genes 72

(Yoshiyama et al 2009 Missirian et al 2014 Ricaud et al 2007) SOG1 induces a 73

robust set of gt100 transcripts by ge4-fold within 15 h in response to 100 Gy IR (Culligan 74

et al 2006 Furukawa et al 2010) SOG1 has a known role in transcriptionally inhibiting 75

cell cycle progression in response to DNA-damage (Preuss and Britt 2003 Yoshiyama et 76

al 2009) The sog1-1 mutant was originally isolated by it lacking the gt6-day growth 77

delay in true leaf development observed during germination in a repair-defective xpf 78

background after a 100 Gy IR dose was applied to imbibed seeds Under these 79

conditions the sog1-1 mutant lacked the DNA damage-induced G2-phase cell cycle 80

arrest observed in its xpf background but the plants were genetically unstable (Preuss and 81

Britt 2003 Huefner et al 2014) The SOG1 transcriptional induction of cell-cycle arrest 82

involves down-regulating certain factors (eg CDKB12 CDKB21 and KNOLLE 83

(Yoshiyama et al 2009 Missirian et al 2014)) whilst directly upregulating other 84

factors (eg CYCB11 SMR-57 (Weimer et al 2016a Yi et al 2014) and WEE1 85


5

indirectly (De Schutter et al 2007 Cools et al 2011)) IR-induced SOG1 also 86

transcriptionally activates DNA repair genes including BRCA1 and RAD51 (Yoshiyama 87

et al 2009) which function in homologous recombination (HR)- based repair in S- and 88

G2-phase during normal growth (Shrivastav et al 2008 Menges et al 2003) SOG1 89

appears to mediate (via upregulated CYCB11 in complex with CDKB1) damage-90

localized HR repair by BRCA1 and RAD51 in conjunction with RBR1 (Biedermann et 91

al 2017 Weimer et al 2016a Horvath et al 2017) HR repair can help to restore a 92

damaged cellrsquos genomic integrity in addition to the faster (Mao et al 2008) 93

predominating canonical non-homologous end-joining repair pathway (Cermak et al 94

2017) SOG1rsquos induction of HR-repair in combination with cell cycle arrest can prevent 95

cell death from mitosis occurring prematurely in the presence of unrepaired DSBs 96

(Furukawa et al 2010 Cools et al 2011 Leguillier et al 2012 Yi et al 2014) 97

98

During normal growth the stem cell niche (SCN) of root and shoot meristems 99

contains stem cells that are maintained in an undifferentiated state Each stem cell can 100

self-renew and produce a transit-amplifying daughter cell in a specialized lsquoasymmetricrsquo 101

cell division The transit-amplifying daughter cells can proliferate through conventional 102

mitotic (symmetrical) cell divisions and subsequently differentiate into specialized cell-103

types The aforementioned processes are regulated by positional signals (Heidstra and 104

Sabatini 2014) Cell type-specific programmed cell death (PCD) has been observed in 105

root meristems of the model plant Arabidopsis after exposure to IR radiomimetic 106

chemicals UV (Curtis and Hays 2007 Furukawa et al 2010 Fulcher and Sablowski 107

2009) and chilling stress (Hong et al 2017) This programmed response requires SOG1 108


6

ATM and (to a lesser degree) ATR as well as de novo protein synthesis (Furukawa et al 109

2010) This PCD is focused in the stele cell initials and their immediate daughters as 110

well as to a lesser extent in columella initials contrastingly in SOG1-deficient lines cell 111

death is observed one day later and is distributed randomly throughout the mitotic 112

population (Furukawa et al 2010) DDR-induced PCD reduces the accumulation of cells 113

with compromised genomic integrity in a multicellular organism (Hu et al 2016) and 114

acts to prevent tumor formation in mammals Most plant species are not susceptible to 115

neoplasia as cells are immobilized by the cell wall and have multifaceted hormone 116

regulation by neighboring cells (Doonan and Sablowski 2010) The relevance of this 117

developmentally-specific DNA damage-induced PCD in the primary root has been 118

somewhat unclear given that the primary rootrsquos function can be effectively replaced by a 119

lateral root and the root does not contribute to the next generation 120

121

Here we report the role of SOG1 in the recovery of the root tip after DNA damage 122

induced by an acute dose of IR (150 Gy) We demonstrate that SOG1-mediated PCD 123

(Furukawa et al 2010) triggers the removal of a subset of stem cells with the resulting 124

cell death triggering a regeneration response in the surrounding root apical meristem 125

(RAM) We demonstrate that SOG1 mediates an arrest to proliferative (anticlinal) cell 126

division which in combination with SOG1rsquos known role in transcriptionally-inducing 127

HR (Yoshiyama et al 2009) likely supports the mitotic-competency of remnant cells 128

We also demonstrate that the regeneration response which involves a partial loss of 129

cellular identities and the induction of regenerative (periclinal) cell divisions facilitates 130

the rebuilding of a functional stem cell niche able to resume proliferative cell division 131


7

(and thus root growth) As SOG1 is unique to seed-bearing plants (Yoshiyama 2016) 132

this developmental response may have evolved to rescue the growth of seed-born 133

embryos from DNA damage accumulated during aging (Waterworth et al 2015) 134

135

136


8

Results 137

Recovery of the primary root after IR involves dissolution and reconstruction of the RAM 138

To understand the recovery of roots after acute DNA damage we followed the 139

long-term effects of an acute dose of IR (150 Gy) on 5-day-old Arabidopsis roots After 140

irradiation growth in WT slowed to less than a millimeter per day for several days (2 ndash 141

5+ d) with growth recovering after about one week sog1 mutants in contrast had 142

stopped all growth by 3 days after IR and did not recover (Fig 1A and B) The difference 143

in growth rate between WT vs sog1-1 after IR was much more noticeable in roots than in 144

shoots (Fig 1A vs Supplemental Figure S1 note that sog1 grows similarly to WT in 145

unstressed conditions) Irradiated WT roots but not sog1 roots undergo short term (6+ h 146

after IR) cell-type-specific induction of PCD in the stele precursor cells and columella 147

initials (Fig 1C) In the days (24+ h) following IR we observed (SOG1-independent) 148

death across the mitotic zone of sog1 roots in all cell types (Fig 1C) whereas such death 149

was not observed across the mitotic zone of WT roots These root cell death patterns are 150

consistent with previous observations of the short-term (le2 d) effects of SOG1 deficiency 151

after 100 Gy IR (Furukawa et al 2010) 152

IR also affected the structure as well as the growth rate of the RAM The 153

Quiescent Center (QC) is a cluster of 4 cells that rarely divide with the stem cells 154

dividing proximal to it driving root elongation and the cells dividing distal to it 155

producing the continually-sloughing root cap (Heidstra and Sabatini 2014) Concurrent 156

with the aforementioned growth arrest in WT (2 ndash 5+ days post IR) the stereotypical 157

pattern of cells in the WT RAM became disorganized and the QC became impossible to 158

identify morphologically (Fig 1C) Over the next 5 days we observed that a QC-specific 159


9marker pWOX5ERGFP (ten Hove et al 2010) transiently expanded its expression in an 160


10irregular pattern that included the former position of the QC (Fig 2A) but later refocused 161


11

as the stereotypical RAM (and root growth) was reestablished This pWOX5ERGFP 162

expansion was previously observed after a chronic (24 h) exposure to a radiomimetic 163

compound (06 microgmL bleomycin) This WOX5 expansion was thought to reflect QC cell 164

division (Heyman et al 2013) consistent with the long-established hypothesis that the 165

QC can divide to replace adjacent stem cells if they die (Clowes 1959) 166

If the expansion of WOX5 expression simply reflects an enlargement of the QC 167

one would expect to see additional QC-specific markers expressed in the same pattern 168

We followed the QC- and SCN-marker pAGL42ERGFP (Nawy et al 2005) and 169

observed a transient expansion of expression then refocusing as RAM organization and 170

root growth were restored (Supplemental Figure S2A) similar to the changes observed 171

for pWOX5ERGFP We then followed the expression of two enhancer-trap GUS markers 172

QC25 (which is expressed in both the QC and the more distal columella cells) and QC46 173

(Sabatini et al 2003) The expression of these markers transiently diminished (in 174

contrast to the expansion observed for WOX5 and AGL42) before being reestablished 175

along with RAM organization and root growth Put together these findings suggest that 176

some aspects of QC identity were lost (Sabatini et al 2003) andor that some aspects of 177

QC identity were ectopically-acquired by neighboring cells (Heyman et al 2013) It is 178

unlikely that the loss of the QC markers was due to the death of QC cells as they are 179

particularly resistant to IR-induced cell death (Curtis and Hays 2007 Heyman et al 180

2013) in contrast to the cells that surround the QC (Fig 1C) (Furukawa et al 2010 181

Yoshiyama et al 2013) 182

Further support for the premise that RAM cells undergo partial loss of identity 183

during the restoration of SCN mitotic-competency came from the rapid and dramatic 184


12

expansion then refocusing of the pCYCD6ERGFP (Sozzani et al 2010) 185

cortexendodermis initial (CEI) cell marker (Supplemental Figure S2B) This gene is 186

involved in promoting the periclinal division of the CEI cell during normal growth in 187

order to form the cortex and endodermal cell files (Heidstra and Sabatini 2014 Sozzani 188

et al 2010) The identity of the stele precursor cells as characterized by pWOLERGFP 189

expression (Birnbaum et al 2003) (Supplemental Figure S2C) remained unchanged 190

despite the expansion of pWOX5ERGFP expression into this stele tissue Localization of 191

the auxin maximum in the QC is crucial for root SCN function (Heidstra and Sabatini 192

2014) and was surveyed based on pDR5ERGFP expression (Ottenschlager et al 2003) 193

We did not observe noticeable changes in this auxin maximum (Supplemental Figure S3) 194

such as were recently described in response to chilling stress (Hong et al 2017) Put 195

together the changes we observed in cell type marker expression suggest that both the 196

QC and its surrounding cells undergo a partial loss of identity during the recovery 197

process 198

199

Dissolution of RAM organization is a programmed response requiring SOG1 200

The QC of WT root tips became morphologically unrecognizable within 48 hours 201

of IR whereas the stereotypical organization of the RAM was stable in sog1 (Fig 1C) In 202

addition expansion of the WOX5 expression domain (2 - 6 d) was observed in WT roots 203

but not in sog1 roots (Fig 2A) We observed that recovery of normal root growth rate and 204

morphology occurred in WT 7 days after IR whereas the sog1 root tip failed to recover 205

and showed cellular enlargements normally only observed in the cells of the elongation 206

zone (Fig 1C) These cellular enlargements in sog1 probably reflected the progression of 207


13

differentiation and endoreduplication (which occurs normally during root development) 208

down to the tip of the arrested root The conservation of root tip structure and cell identity 209

in sog1 suggests that SOG1-dependent PCD (andor another unknown SOG1-dependent 210

response) is associated with the disrupted RAM identity displayed in irradiated WT 211

plants Root growth recovery is thus associated with both the PCD and partial loss of cell-212

type identities observed in WT root tips both of these processes are absent in sog1 root 213

tips 214

215

SOG1 is required for cell cycle arrest immediately after IR 216

In sog1 mutants we observed (SOG1-independent) death in the days following IR 217

including more cell death across the mitotic zone (in all cell-types without a SCN-focus) 218

compared with WT (eg Figure 2A WT vs sog1 at 32 h after IR) We hypothesized that 219

this death in sog1 may be due to mitosis proceeding in the presence of unrepaired DSBs 220

based on the distribution across the mitotic zone of the root and the delayed onset with 221

respect to PCD (32 h vs 8 h) (Yi et al 2014 Furukawa et al 2010 Cools et al 2011) 222

To investigate the role of SOG1 (and hence the transcriptional response) in cell cycle 223

arrest after IR we measured DNA replication (as EdU incorporation during S-phase) and 224

cell division (by visualizing metaphase cells using a fluorescent histone marker) in WT 225

and sog1 lines We found that DNA replication (Fig 3A) and cell division (Fig 3B) 226

were largely inhibited in the mitotic zone 6 - 10 h after IR in WT seedlings but not in 227

sog1 The lack of cell cycle control in sog1 coupled with the defective induction of HR 228

repair transcripts (Yoshiyama et al 2009) may be responsible for the presumably 229

unprogrammed cell death in the mitotic zone (Furukawa et al 2010) We also wished to 230


14

analyze the frequency of regenerative periclinal (sideways) cell divisions which establish 231

new cell files and thus are essential for the restoration of a functional root meristem 232


15

(Heyman et al 2016) during growth recovery As part of visualizing metaphase cells 233

we found an average of 1 - 2 periclinal metaphase cells per WT root but essentially none 234

per sog1 root from 5 - 7 d after IR (Fig 3B) Furthermore by 5 d after IR WT roots had 235

restored their proliferative anticlinal (lengthwise) cell divisions which contribute to root 236

elongation (Heidstra and Sabatini 2014) to levels observed in mock-irradiated roots 237

238

ERF115 is induced ectopically outside of the QC in response to cell death 239

ETHYLENE RESPONSE FACTOR115 (ERF115 AT5G07310) is a gene recently 240

demonstrated to be induced in cells that are near dead cells in the root tip 241

(Heyman et al 2016 Zhang et al 2016) Along with PHYTOCHROME A SIGNAL 242

TRANSDUCTION1 PAT1) ERF115 has a key role in promoting regenerative divisions 243

of neighboring cells and thus is important in the maintenance and recovery of a damaged 244

SCN (Heyman et al 2013 Heyman et al 2016) We sought to investigate the induction 245

of ERF115 alongside PCD after IR Accordingly we visualized the pERF115NLS(-246

GUS)GFP marker line (Heyman et al 2013) hourly during the onset of PCD (3 - 7 h 247

after IR) while staining for cell death We found that ERF115 was induced in the same 248

5+ h time-frame and cell types as PCD in WT roots (Fig 4) We found that ERF115 249

expression was induced in an average of 2 - 3 remnant cells per each dead cell as 250

observable in these two-dimensional images (6 and 7 h after IR Fig 4) 251

252

To determine the duration of ERF115 induction after IR we visualized the 253

pERF115GFPERF115 marker line that encodes the ERF115 protein (Heyman et al 254

2013) which is particularly subject to degradation in the unperturbed cell (Heyman et al 255


16

2013) We found that pERF115GFPERF115 expression peaked a few days after IR and 256

disappeared once root growth was restored (Supplemental Figure S4) In these 257

visualizations we could clearly observe ERF115 expression focused in the stele 258

precursor cells 259

260

We then sought to determine the dependency of ERF115 induction on SOG1 At 8 h after 261

IR we found that ERF115 was specifically induced in WT roots in a SCN-focused 262

pattern similar to the induction of PCD in WT (Fig 5A B) In sog1 roots the focused 263

expression in the SCN is lost there is instead ERF115 induction across the mitotic zone 264

at 32 h after IR located within the stele and to a limited extent in the endodermis (Fig 265


17

5A) Cell death is similarly present in these stele and endodermal tissues in sog1 lines 266

(Fig 5B) The cortical and epidermal cells in the mitotic zone of sog1 lines also exhibit 267

cell death yet no ERF115 expression this finding is in line with a previous study that 268


18

found these cell types did not induce ERF115 or regenerative periclinal cell divisions in 269

response to dead cells nearby (Heyman et al 2016) This ERF115 marker was also 270

induced in the shoot similar to that seen in the root with a SOG1-dependent 8 h-onset 271

and focus in the shoot apical meristem for WT (Supplemental Figure S5) In the sog1 272

mutant the 8 h-induction focused in the shoot apical meristem was lost and ERF115 273

expression was observed at 32 h after IR throughout the growing leaflet 274

275

The results described above for the root in which cell death is easily assayed indicate 276

that the induction of ERF115 can be induced by cell death in the absence SOG1 and that 277

it roughly follows the pattern of cell death It has been previously shown that ERF115 278

expression has been induced in WT upon wounding roots by excising their tips (Heyman 279

et al 2016) To determine whether this ERF115 induction was also SOG1-independent 280

we compared wound-induced pERF115GUS expression in WT vs sog1 roots We found 281

that pERF115GUS was indeed induced by death regardless of the presence or absence 282

of SOG1 (Fig 5C) In the decapitated root we observed ERF115-induction primarily in 283

stele cells which play an important role in root tip regeneration (Efroni et al 2016) 284

Similar to a previous study (Heyman et al 2016) we did not see ERF115 induction in 285

epidermal and cortical cells bordering the stump of the decapitated root Taken together 286

these observations indicate that pERF115GUS expression is driven by cell death be it 287

from SOG1-dependent PCD from SOG1-independent mitosis-linked cell death or from 288

wounding-induced death It is possible that the induction of ERF115 by IR in the SCN is 289

the one exception to this rule and does require transcriptional induction by SOG1 but the 290


19

simpler hypothesis is that SOG1 is instead inducing death and death per se is inducing 291

ERF115 292

293

The transcriptional induction of ERF115 has not been previously observed in IR-294

response transcriptomes (Ricaud et al 2007 Yoshiyama et al 2009 Missirian et al 295

2014) possibly due to its localized expression in a small subset of cells Nonetheless this 296

discrepancy did raise the question of whether its IR-induction is an artifact of the GUS or 297

GFP fusion transgenes rather than the native gene To follow expression of the native 298

gene we employed cell-type specific transcriptomics A pWOLERGFP line (Birnbaum et 299

al 2003) was used to purify GFP+ protoplasts from the stele precursor cells We would 300

not expect to capture QC cells when purifying for cells expressing this construct (Efroni 301

et al 2015 Efroni et al 2016) We could thus compare the transcriptomes of irradiated 302

stele precursor cells (during the onset of PCD 85 h after 100 Gy Supplemental Figures 303

S6 and S7) vs entire root tips ERF115 was indeed transcriptionally induced in the stele 304

precursors (Fig 6) We found that ERF114 (ERF115rsquos closest homolog AT5G61890) 305

and PAT1 (ERF115rsquos partner) were also induced in stele precursors during the onset of 306

PCD (Fig 6) 307

We investigated the role of ERF115 in root growth recovery We found that 308

erf115 mutant seedlings could recover their growth after IR albeit with a delay of several 309

days with respect to WT (Supplemental Figure S8) Considering the apparent functional 310

redundancy of ERF115 in its initially reported role in promoting QC cell division 311

(Heyman et al 2013) we then analyzed the p35SERF115-SRDX transgenic line 312

(Heyman et al 2013 Heyman et al 2016) Using this line which expresses a dominant 313


20

transcriptional repressor of ERF115rsquos targets we found that the majority of roots failed to 314

recover their growth (Supplemental Figure S9) even when analyzed over a longer (15 d) 315

time-course It was previously reported that p35SERF115-SRDX transgenics failed to 316

recover growth over a shorter (5 d) time course which was done after a chronic (24 h) 317

exposure to (06 microgmL) bleomycin (Heyman et al 2013) 318

319

320


21

Discussion 321

SOG1 is required to direct an ERF115-dependent regeneration pathway and an 322

immediate cell cycle arrest in response to IR 323

Here we describe key roles of SOG1 in the long-term growth recovery of the Arabidopsis 324

seedlingrsquos primary root after acute DNA damage WT primary roots but not those of 325

sog1 mutants can recover their growth after a severe (150 Gy) IR dose after 7 days This 326

recovery requires an initial SOG1-mediated DDR which serves in the long-term 327

regeneration of a mitotically-competent SCN Our prior understanding of SOG1 in the 328

DDR has been essentially limited to short-term effects namely the transcriptional 329

induction of PCD (Furukawa et al 2010 Yoshiyama et al 2009) cell cycle inhibitors 330

(Culligan et al 2006 Yoshiyama et al 2009 Yi et al 2014) and DNA repair factors 331

(Yoshiyama et al 2009) 332

333

SOG1 transcriptionally regulates a variety of short-term (le 2 d) processes in the DDR as 334

described following SOG1 induces PCD in many of the IR-damaged stem cells (and 335

their daughters) in the first 6 - 10 hours after IR (Furukawa et al 2010) thereby rapidly 336

but indirectly inducing an ERF115-mediated regeneration response near the damaged 337

SCN sog1rsquos defect in PCD in the IR-compromised SCN (6 - 10 h after IR) results in the 338

persistence of damaged cells which might then act as an anatomical block to 339

regeneration We also demonstrate that SOG1 is required for the arrest of the cell cycle in 340

these surviving cells also within 6 - 10 hours after IR This arrest along with the 341

previously-reported role of SOG1 in the induction DNA repair transcripts (Culligan et al 342

2006 Yoshiyama et al 2013) can support the mitotic competency of remnant cells that 343


22

are needed to replenish the IR-compromised SCN and hence resume growth sog1 344

mutantsrsquo defect in cell cycle arrest (8 h) along with their failed induction of DNA repair 345

transcripts (Yoshiyama et al 2013) predisposes them to the (32 h onset) cell death seen 346

across the rootrsquos mitotic zone in all cell types A similar (patchy) pattern of IR-induced 347

cell death across the mitotic zone was also demonstrated in atr and atr atm double 348

mutants (Furukawa et al 2010) ERF115 induction is associated with the cell death 349

across the mitotic zone (primarily in the stele tissue) in sog1 roots (Fig 5A B) however 350

ERF115 induction in the SCN is weak in sog1 relative to WT ERF115 induction is 351

associated with cell death induced by wounding in WT (Heyman et al 2016) and in sog1 352

(Fig 5C) Both of these experiments show that ERF115 is induced by cell death 353

(independently of SOG1) and hence strongly suggest that the SCN-focused ERF115-354

induction in WT roots after IR is due to SOG1-dependent PCD rather than due to a more 355

direct transcriptional induction by SOG1 356

357

We have observed two long-term processes associated with SOG1 activity and root 358

growth recovery after IR The first process is the transient loss of the stereotypical RAM 359

structure including some loss in identity for its constituent cell types (2 - 5 d after IR) 360

which subsequently reform along with the restoration of proliferative cell divisions 361

responsible for growth (5+ d after IR) The second process we have observed in root 362

growth recovery is the occurrence of regenerative periclinal cell division in most WT 363

roots (5 - 7 d after IR) Such cell division may function in replacing the (re)growth-364

enabling SCN cells from mitotically-competent remnant cells nearby (Heyman et al 365

2016) such as the transit-amplifying cells (Efroni et al 2016) andor surviving SCN 366


23

cells We reason that these changes in cytoarchitecture occur as a response to SOG1-367

dependent PCD at the SCN as such cell identity changes have been previously observed 368

during regeneration after cell ablation (van den Berg et al 1997) or root tip excision 369

(Efroni et al 2016) The process of SCN regeneration after SOG1-dependent PCD may 370

like SCN regeneration after excision follow the plantrsquos endogenous positional patterning 371

that is established in the embryo (Efroni et al 2016) sog1 mutants in contrast maintain 372

a stereotypical RAM including a well-defined QC surrounded by a mitotically-373

compromised SCN rather than undergoing the partial loss of cellular identities (2 - 5 d) 374

observed in WT Moreover sog1 mutants fail to induce the regenerative periclinal cell 375

divisions (5 - 7 d after IR) that seem necessary to reform a mitotically-competent SCN 376

only 1 periclinal cell division was observed across 24 sog1 roots whereas 30 such 377

divisions were observed across 24 WT roots The failure of the aforementioned SOG1-378

dependent processes likely contributes to the permanent growth arrest and terminal 379

differentiation observed in sog1 roots Put together we conclude that SOG1 functions in 380

salvaging the overall mitotic competency of the primary root after IR both by removing 381

damage-compromised SCN cells to stimulate (normally very rare) periclinal cell 382

divisions for replacing these dead cells as well as inducing cell cycle arrest and DNA 383

repair in remnant cells 384

385

What role might this SOG1-dependent PCD have during normal plant development 386

We have observed that the Arabidopsis primary root will undergo a week-long process of 387

growth restoration in response to acute DNA damage even though the rootrsquos growth 388

might be restored via the production of a lateral root During a plantrsquos normal growth in 389


24

the soil a growing root tip might encounter a persistent source of DNA damage such as 390

from the presence of a toxic heavy metal (Hu et al 2016 Sjogren et al 2015) In this 391

scenario the preferential growth of lateral roots (rather than a futile cycle of primary root 392

DDR-induced PCD and regeneration) could successfully redirect root growth away from 393

the chronic DNA damaging agent In contrast the restoration of primary root tip growth 394

seems most cost-beneficial (and evolutionarily-favorable) in the case of an acute and 395

transient exposure to DNA damage which is the case for seeds upon imbibition where 396

they acutely experience the DNA damage they accumulated during aging (Waterworth et 397

al 2016 Waterworth et al 2015) Regeneration of the embryonic root in response to 398

DNA damage is critical for the viability of a germinating seed as the linear-growing 399

embryonic root carries no additional root primordia (Van Norman et al 2013) We 400

propose that SOG1 a gene unique to seed-bearing plants (Yoshiyama 2016) may have 401

evolved to salvage the overall mitotic competency (prevent permanent mitotic arrest) of 402

the embryonic root during the germination of aged seeds 403

404

What are the relative contributions of individual DDR and regeneration processes to the 405

overall root growth recovery 406

Some stem cells and some of their early descendants are more prone to IR-induced PCD 407

than others The factors that specifically regulate cell-type specific PCD downstream of 408

SOG1 are unknown (Hu et al 2016) It is possible that damaged cells begin PCD after 409

reaching a critical threshold of damage and that threshold may depend on both the cell 410

type and its position in cell cycle (Hu et al 2016) 411

412


25

Due to the absence of lines uniquely defective in PCD (but not in other SOG1-influenced 413

processes (Hu et al 2016)) we have not been able to characterize the unique 414

contribution of PCD to the growth recovery of the damaged root relative to the 415

contribution of other SOG1-induced processes (eg cell cycle arrest and DNA repair) 416

Nonetheless it is likely that various SOG1-induced genes contribute to root growth 417

recovery after DNA-damage which can be appreciated by the hypersensitivity of relevant 418

mutants to DNA damage For example the growth of cycb11 and rad51 mutants is 419

compromised in response to cisplatin a DNA crosslinker and DSB-inducer (Weimer et 420

al 2016a) Similarly brca1 mutants show enhanced cell death to MMC a DNA 421

crosslinker (Horvath et al 2017) DNA damage-induced hypersensitivity was also 422

observed for mutants in PARP-12 (Song et al 2015) and RAD17 (Heitzeberg et al 423

2004) in response to bleomycin and MMC these genes are also damage-induced by 424

SOG1 (Culligan et al 2006) with a function in an alternative microhomology-mediated 425

NHEJ repair pathway and in ssDNA-sensing for checkpoint control respectively 426

(Shrivastav et al 2008 Hu et al 2016) 427

428

It is possible that the transient enlargement of the zone of expression for the 429

pCYCD6ERGFP (Sozzani et al 2010) marker of cortexendodermis initial cells 430

(Supplemental Figure S2B 8 h ndash 5 d) is due to its role in the promotion of periclinal cell 431

division which CEI cells undergo during normal growth to form the cortex and 432

endodermal cell files (Heidstra and Sabatini 2014 Sozzani et al 2010) The expansion 433

of this marker into a variety cells within the RAM may reflect the replacement of dead 434

cells by replenishing periclinal divisions of neighboring cells (Heyman et al 2016) The 435


26

finding that p35SERF115-SRDX plants generally failed to recover their growth after 436

damage (Heyman et al 2013) whereas erf115 knockout mutants merely exhibited a 437

delayed restoration of growth suggest that there is an alternate ldquobackuprdquo pathway for 438

SCN regeneration It is possible that ERF114 plays a role in this alternative pathway for 439

regeneration as it is the closest homolog of ERF115 (Heyman et al 2016) and is also 440

IR-inducible in the stele progenitor cells (Fig 6) It is also possible that an alternate 441

regeneration pathway may be involved 442

443

Materials and Methods 444

Growth and irradiation of seedlings Arabidopsis thaliana seeds were sterilized in 20 445

Clorox Bleach 01 Triton X-100 and sown on 1x MS salts 03 sucrose (Sigma) 446

08 Phytoagar (BioWorld) pH 57 Seedlings were grown on vertical plates in 16 h 447

days under cool white lamps (photon flux density of 100 μmol mminus2 sminus1) 20˚C for 5 days 448

after having stratified for 48 hours at 4˚C Plants were restored to these conditions after 449

IR or cutting Any time point specified in text is the amount of time that has passed after 450

completion of IR or cutting The seedlings were gamma-irradiated in the dark using a 451

Cs137 source with a dose rate of either 595 Gymin (for the tissue-specific 452

transcriptomics experiment) or 18 Gymin for all other experiments Irradiations were 453

performed between 8 - 10 am with typical experiments requiring a 15 h exposure The 454

growth chamber lamps are on from 8 am - midnight 455

Plant material and transgenic lines We employed Arabidopsis thaliana Col-0 456

(Columbia) as WT The sog1-1 line was derived from our previously reported xpf-2 sog1-457

1 cycB11GUS line a Landsberg erectaCol hybrid (Preuss and Britt 2003) by 458


27

backcrossing to Col-0 twice and then self-pollinated to generate homozygous sog1-1 459

lines sog1-9 was derived from a tetraploid Col-0 TILLING population (Tsai et al 2013) 460

and haploidized via GFP-tailswap(-CENH3) (Ravi et al 2014) The resulting diploid was 461

backcrossed three times to Col-0 and then self-pollinated to generate homozygotes sog1-462

9 carries a nonsense C-to-T mutation 826 bp downstream of the ATG The 463

pERF115NLS-GUSGFP (Heyman et al 2013) and pWOX5ERGFP (ten Hove et al 464

2010 Blilou et al 2005) markers were each crossed with sog1-1 and sog1-9 lines plants 465

that were homozygous for the sog1 alleles were identified in the F2 Experiments were 466

performed with sog1 lines that were segregating for the markers scoring only those 467

seedlings expressing the marker in lateral andor primary roots Similarly the 468

pRPS5aH2BeCFP marker line was crossed with sog1-9 mutant homozygotes were 469

identified in the F2 then used for mitotic figure experiments (along with the 470

pRPS5aH2BeCFP marker line) A studentrsquos t-test was applied to the comparisons 471

shown in Fig 1A and Fig 3B with two tails and the assumption of unequal variance 472

GUS staining and visualization Seedlings were harvested into 04 mL of ice-cold 80 473

acetone in a 48 well flat-bottom Costar microtitre plate (Corning) and incubated at room 474

temperature for 20 m Following removal of the acetone the samples were incubated in 475

02 mL of GUS staining buffer (25 mM NaH2PO4Na2HPO4 buffer (pH 70) 5 mM 476

K3Fe(CN)6 5 mM K4Fe(CN)6 025 Triton 025 mM EDTA 1 mgmL X-Gluc Gold 477

Biotechnology Inc) at 37degC for 1 h The samples were mounted on slides in an 831 478

mixture of chloral hydratewaterglycerol and analyzed using an Axioskop 2 plus 479

microscope (Zeiss) under DIC optics using the Axiovision program (version 48) 480


28

Tissue-specific transcriptomics of cell death Five-day-old pWOLERGFP (Birnbaum et 481

al 2003) seedlings grown on 08 MS + 1 sucrose media were irradiated to 100 Gy at 482

a dose rate of 594 Gymin in the dark Fifteen minutes after IR plates were returned to 483

the growth chamber Five-and-a-half hours after IR root tips were chopped and 484

protoplasted as described (Birnbaum et al 2005) Protoplasts were sorted with a 485

Cytomation MoFlo Cell Sorter and frozen in liquid nitrogen ~3 h after chopping RNA 486

was isolated using Trizol and mRNA was captured using OligodT Dynabeads using the 487

manufacturerrsquos protocol (Invitrogen) RNA-seq libraries were created as described 488

(Kumar et al 2012) with the modification that the total RNA was extracted and purified 489

from the protoplasts using Trizol The libraries were multiplexed and sequenced using 490

Illuminarsquos GAII and HiSeq 2000 Reads were quality trimmed with a custom script 491

(written by Ted Toal UC Davis) Alignment to TAIR10 was performed with BWA-492

MEM (Li 2013) transcript abundance was counted using the HTSeq library (Anders et 493

al 2014) and differential expression calls were calculated with DEseq2 (Love et al 494

2014) v145 Raw data is available in the SRA with the BioProject Accession 495

PRJNA380494 496

Visualization of cell death Seedlings were incubated with 5 μgmL propidium iodide in 497

water for 5 m on a microscope slide before confocal imaging with a Zeiss LSM710 498

EdU incorporation Using the Click-iTreg EdU Alexa Fluorreg 488 Imaging Kit 499

(Invitrogen) seedlings were incubated with 10 microM EdU for 4 hours to measure S-phase 500

entry beginning at 6 24 and 48 h after IR or mock treatment before confocal imaging 501

Nuclei were counted in the mitotic zone using Image J with the plugin ITCN (width 15 502

min distance 75 threshold 01) 503


29

Tip excision pERF115NLS-GUSGFP primary roots were cut by hand with an 18 Ga x 504

1rsquorsquo Monoject 200 needle (Medtronic) in the approximate QC position After incubation 505

the seedlings were stained for GUS activity and visualized 506

507

Supplementary Materials 508

Figure S1 True leaf production after IR 509

Figure S2 Effects of IR on cell type-specific marker expression 510

Figure S3 Effects of IR on pDR5ERGFP expression 511

Figure S4 Effects of IR on pERF115GFPERF115 expression 512

Figure S5 pERF115GUS expression in the shoot apical meristem 513

Figure S6 Representation of roots used to obtain WOL marker-expressing cells vs 514

whole root tip cells for stele-specific transcriptomics 515

Figure S7 Timing of PCD after 100 Gy IR 516

Figure S8 Growth recovery after IR in erf115-- 517

Figure S9 Growth recovery after IR in p35SERF115-SRDX 518

519

Accession Numbers 520

ERF114 (AT5G61890) ERF115 (AT5G07310) PAT1 (AT5G48150) and SOG1 521

(AT1G25580) 522

523

Acknowledgements 524

We thank Lieven De Veylder (VIB and U Ghent) for pERF115NLS-GUSGFP 525

pERF115ERF115GFP erf115 (KO SALK_021981) and p35SERF115-SRDX Philip 526


30

Benfey (Duke U) for pWOLERGFP Mohan Marimuthu and Luca Comai (UC Davis) for 527

developing and sharing their unpublished pRPS5aH2BeCFP marker line Elliot 528

Meyerowitz (CalTech) for pWOX5ERGFP Renze Heidstra (Wageningen U) for the 529

GUS enhancer trap lines QC25 and QC46 Klaus Palme (U Freiburg) for the 530

pDR5ERGFP marker line Natalie Clark and Ross Sozzani (North Carolina State U) for 531

other GFP lines and Idan Efroni (the Hebrew U) for root cutting advice 532

533

Figure Legends 534

Figure 1 Growth recovery after IR A Root growth rate per day (growth since 535

previous day) after 150 Gy IR as a fraction of root growth rate for the mock-irradiated 536

control in WT sog1-1 and sog1-1 + tgSOG1 sog1-1 samples are significantly 537

different from WT (p-value lt 0001) WT samples at 7 days after IR are significantly 538

different from 6 days after IR (p-value lt 005) B WT (left) and sog1-1 (right) roots at 8 539

days after IR Arrowhead indicates root tip position at time of IR C Five-day-old WT 540

sog1-1 or sog1-1 + SOG1 root tips were stained with propidium iodide and imaged up to 541

7 days after 150 Gy IR 542

543

Figure 2 Effects of IR on QC marker expression A Five-day-old seedlings carrying 544

pWOX5ERGFP in WT sog1-1 or sog1-9 backgrounds were irradiated and imaged up to 8 545

days after 150 Gy IR False color black = propidium iodide purple = GFP B Five-day-546

old seedlings carrying QC25 (top) or QC46 (bottom) GUS enhancer trap lines were 547

stained and imaged up to 8 days after 150 Gy IR 548

549


31

Figure 3 Effects of SOG1 on cell cycle arrest in the RAM after irradiation 550

A DNA replication in the mitotic zone measured as EdU labeled nuclei 5-day-old 551

seedlings were irradiated to 150 Gy and labeled with EdU for 4 h beginning at 6 h after 552

the completion of IR Error bars are standard error (WT N = 9 sog1-1 N = 6 sog1-1 + 553

tgSOG1 N = 6 WT +IR N = 11 sog1-1 +IR N = 10 sog1-1 + tgSOG1 +IR N = 10) + 554

IR samples are significantly different from mock-IR controls (p-value lt 005) B The no 555

of metaphase cells per root tip observed in anticlinal vs periclinal orientations up to 7 556

days after 5-day-old WT or sog1-9 seedlings both carrying pRPS5aH2BeCFP were 557

irradiated with 150 Gy IR Error bars are standard error (WT N = 8 sog1-9 N = 8) and 558

sog1-9 samples are significantly different from WT (p-values are lt 005 and lt 001 559

respectively) 560

561

Figure 4 Effects of IR on ERF115 expression and PCD Five-day-old seedlings carrying 562

pERF115NLS-GUSGFP in WT backgrounds were irradiated and imaged up to 7 hours 563

after 150 Gy IR False color black = propidium iodide purple = GFP 564

565

Figure 5 pERF115GUS expression after cell death A Five-day-old seedlings carrying 566

pERF115NLS-GUSGFP were irradiated and imaged 8 and 32 hours after 150 Gy IR (or 567

mock irradiation) B Five-day-old seedlings were irradiated with 150 Gy Seedlings were 568

stained with Propidium Iodide (PI) to visualize PCD up to 32 hours after IR C Five-day-569

old WT or sog1-1 seedlings carrying pERF115NLS-GUSGFP were either cut in the 570

meristematic zone or left intact then stained with PI (top) up to 8 hours after cutting and 571


32

immediately following visualization the same seedlings were stained for GUS activity 572

(bottom) 573

574

Figure 6 WOX5 ERF115 induction during the onset of PCD WOX5 ERF115 ERF114 575

PAT1 ACT1 (ACTIN1) fold change in GFP+ (stele-specific) or whole root tip protoplasts 576

during the onset of PCD after 100 Gy IR Error bars are standard error Irradiated 577

samples are significantly differentially expressed from mock-irradiated controls (p-value 578

lt 005) 579

580

581


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BIRNBAUM K JUNG J W WANG J Y LAMBERT G M HIRST J A GALBRAITH D W amp BENFEY P N 2005 Cell type-specificexpression profiling in plants via cell sorting of protoplasts from fluorescent reporter lines Nat Meth 2 615-619


BIRNBAUM K SHASHA D E WANG J Y JUNG J W LAMBERT G M GALBRAITH D W amp BENFEY P N 2003 A geneexpression map of the Arabidopsis root Science 302 1956-60


BLILOU I XU J WILDWATER M WILLEMSEN V PAPONOV I FRIML J HEIDSTRA R AIDA M PALME K amp SCHERES B 2005The PIN auxin efflux facilitator network controls growth and patterning in Arabidopsis roots Nature 433 39-44


CERMAK T CURTIN S J GIL-HUMANES J CEGAN R KONO T J Y KONECNA E BELANTO J J STARKER C G MATHREJ W GREENSTEIN R L amp VOYTAS D F 2017 A Multipurpose Toolkit to Enable Advanced Genome Engineering in Plants Plant Cell29 1196-1217


CLOWES F A L 1959 Reorganization of root apices after irradiation Annals of Botany 23 205-210Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

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CULLIGAN K M ROBERTSON C E FOREMAN J DOERNER P amp BRITT A B 2006 ATR and ATM play both distinct and additiveroles in response to ionizing radiation Plant J 48 947-61


CURTIS M J amp HAYS J B 2007 Tolerance of dividing cells to replication stress in UVB-irradiated Arabidopsis roots requirementsfor DNA translesion polymerases eta and zeta DNA Repair (Amst) 6 1341-58


DE SCHUTTER K JOUBES J COOLS T VERKEST A CORELLOU F BABIYCHUK E VAN DER SCHUEREN E BEECKMAN TKUSHNIR S INZE D amp DE VEYLDER L 2007 Arabidopsis WEE1 kinase controls cell cycle arrest in response to activation of the DNAintegrity checkpoint Plant Cell 19 211-25

Pubmed Author and TitleCrossRef Author and Title wwwplantphysiolorgon May 26 2020 - Published by Downloaded from

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DOONAN J H amp SABLOWSKI R 2010 Walls around tumours mdash why plants do not develop cancer Nat Rev Cancer 10 794-802Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

EFRONI I IP P-L NAWY T MELLO A amp BIRNBAUM K D 2015 Quantification of cell identity from single-cell gene expressionprofiles Genome Biology 16 9


EFRONI I MELLO A NAWY T IP P L RAHNI R DELROSE N POWERS A SATIJA R amp BIRNBAUM K D 2016 RootRegeneration Triggers an Embryo-like Sequence Guided by Hormonal Interactions Cell 165 1721-1733


EINSET J amp COLLINS A R 2015 DNA repair after X-irradiation lessons from plants Mutagenesis 30 45-50Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

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FURUKAWA T CURTIS M J TOMINEY C M DUONG Y H WILCOX B W AGGOUNE D HAYS J B amp BRITT A B 2010 Ashared DNA-damage-response pathway for induction of stem-cell death by UVB and by gamma irradiation DNA Repair (Amst) 9 940-8


HEIDSTRA R amp SABATINI S 2014 Plant and animal stem cells similar yet different Nat Rev Mol Cell Biol 15 301-12Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

HEITZEBERG F CHEN I P HARTUNG F OREL N ANGELIS K J amp PUCHTA H 2004 The Rad17 homologue of Arabidopsis isinvolved in the regulation of DNA damage repair and homologous recombination Plant J 38 954-68


HEYMAN J COOLS T CANHER B SHAVIALENKA S TRAAS J VERCAUTEREN I VAN DEN DAELE H PERSIAU G DEJAEGER G SUGIMOTO K amp DE VEYLDER L 2016 The heterodimeric transcription factor complex ERF115ndashPAT1 grantsregeneration competence Nature Plants 2 16165


HEYMAN J COOLS T VANDENBUSSCHE F HEYNDRICKX K S VAN LEENE J VERCAUTEREN I VANDERAUWERA SVANDEPOELE K DE JAEGER G VAN DER STRAETEN D amp DE VEYLDER L 2013 ERF115 controls root quiescent center celldivision and stem cell replenishment Science 342 860-3


HONG J H SAVINA M DU J DEVENDRAN A KANNIVADI RAMAKANTH K TIAN X SIM W S MIRONOVA V V amp XU J 2017 ASacrifice-for-Survival Mechanism Protects Root Stem Cell Niche from Chilling Stress Cell 170 102-113e14


HORVATH B M KOUROVA H NAGY S NEMETH E MAGYAR Z PAPDI C AHMAD Z SANCHEZ-PEREZ G F PERILLI SBLILOU I PETTKO-SZANDTNER A DARULA Z MESZAROS T BINAROVA P BOGRE L amp SCHERES B 2017 ArabidopsisRETINOBLASTOMA RELATED directly regulates DNA damage responses through functions beyond cell cycle control Embo j 361261-1278


HU Z COOLS T amp DE VEYLDER L 2016 Mechanisms Used by Plants to Cope with DNA Damage Annu Rev Plant Biol 67 439-62 wwwplantphysiolorgon May 26 2020 - Published by Downloaded from



HUEFNER N D YOSHIYAMA K FRIESNER J D CONKLIN P A amp BRITT A B 2014 Genomic stability in response to high versuslow linear energy transfer radiation in Arabidopsis thaliana Front Plant Sci 5 206


KUMAR R ICHIHASHI Y KIMURA S CHITWOOD D H HEADLAND L R PENG J MALOOF J N amp SINHA N R 2012 A High-Throughput Method for Illumina RNA-Seq Library Preparation Front Plant Sci 3 202


LEGUILLIER T VANDORMAEL-POURNIN S ARTUS J HOULARD M PICARD C BERNEX F ROBINE S amp COHEN-TANNOUDJIM 2012 Omcg1 is critically required for mitosis in rapidly dividing mouse intestinal progenitors and embryonic stem cells Biol Open 1648-57


LI H 2013 Aligning sequence reads clone sequences and assembly contigs with BWA-MEM Preprint at arXivhttparxivorgabs13033997v2


LOVE M I HUBER W amp ANDERS S 2014 Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2Genome Biol 15 550


MAO Z BOZZELLA M SELUANOV A amp GORBUNOVA V 2008 Comparison of nonhomologous end joining and homologousrecombination in human cells DNA Repair (Amst) 7 1765-71


MENGES M HENNIG L GRUISSEM W amp MURRAY J A 2003 Genome-wide gene expression in an Arabidopsis cell suspensionPlant Mol Biol 53 423-42


MISSIRIAN V CONKLIN P A CULLIGAN K M HUEFNER N D amp BRITT A B 2014 High atomic weight high-energy radiation(HZE) induces transcriptional responses shared with conventional stresses in addition to a core DSB response specific toclastogenic treatments Front Plant Sci 5 364


MOISEENKO V V WAKER A J HAMM R N amp PRESTWICH W V 2001 Calculation of radiation-induced DNA damage from photonsand tritium beta-particles Radiation and Environmental Biophysics 40 33-38


NAWY T LEE J-Y COLINAS J WANG J Y THONGROD S C MALAMY J E BIRNBAUM K amp BENFEY P N 2005Transcriptional Profile of the Arabidopsis Root Quiescent Center The Plant Cell 17 1908-1925


OTTENSCHLAGER I WOLFF P WOLVERTON C BHALERAO R P SANDBERG G ISHIKAWA H EVANS M amp PALME K 2003Gravity-regulated differential auxin transport from columella to lateral root cap cells Proceedings of the National Academy of Sciencesof the United States of America 100 2987-2991


PREUSS S B amp BRITT A B 2003 A DNA-damage-induced cell cycle checkpoint in Arabidopsis Genetics 164 323-34Pubmed Author and Title


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RICAUD L PROUX C RENOU J P PICHON O FOCHESATO S ORTET P amp MONTANE M H 2007 ATM-mediatedtranscriptional and developmental responses to gamma-rays in Arabidopsis PLoS One 2 e430


ROITINGER E HOFER M KOCHER T PICHLER P NOVATCHKOVA M YANG J SCHLOGELHOFER P amp MECHTLER K 2015Quantitative phosphoproteomics of the ataxia telangiectasia-mutated (ATM) and ataxia telangiectasia-mutated and rad3-related (ATR)dependent DNA damage response in Arabidopsis thaliana Mol Cell Proteomics 14 556-71


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Parsed Citations

Reviewer PDF

Parsed Citations

2

Abstract 25

In Arabidopsis DNA damage-induced programmed cell death is limited to the 26

meristematic stem cell niche and its early descendants The significance of this cell-type 27

specific programmed cell death is unclear Here we demonstrate in roots that it is the 28

programmed destruction of the mitotically-compromised stem cell niche that triggers its 29

regeneration enabling growth recovery In contrast to wild-type plants sog1 plants 30

which are defective in damage-induced programmed cell death maintain the cell 31

identities and stereotypical structure of the stem cell niche after irradiation but these cells 32

fail to undergo cell division terminating root growth We propose DNA damage-induced 33

programmed cell death is employed by plants as a developmental response contrasting 34

with its role as an anti-carcinogenic response in animals This role in plants may have 35

evolved to restore the growth of embryos after the accumulation of DNA damage in 36

seeds 37

Keywords Programmed cell death ionizing radiation double-strand breaks stem 38

cell niche 39

40


3

Introduction 41























4



65






















5













98












6













121












7




135

136


8

Results 137




























11

























12














process 198

199










13







tips 214

215

















14




15






238














252





16




precursor cells 259

260







17





18







275

















19


ERF115 292

293














PCD (Fig 6) 307








20






319

320


21

Discussion 321












333












22














357











23



















385






24















404








412


25
















428









26








443

















27
























28
















PRJNA380494 496









29




507












519



(AT1G25580) 522

523





30







533

Figure Legends 534









543






549


31











respectively) 560

561




565








32


(bottom) 573

574





lt 005) 579

580

581























































































































Parsed Citations

Reviewer PDF

Parsed Citations

3

Introduction 41























4



65






















5













98












6













121












7




135

136


8

Results 137




























11

























12














process 198

199










13







tips 214

215

















14




15






238














252





16




precursor cells 259

260







17





18







275

















19


ERF115 292

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319

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Parsed Citations

4



65






















5













98












6













121












7




135

136


8

Results 137




























11

























12














process 198

199










13







tips 214

215

















14




15






238














252





16




precursor cells 259

260







17





18







275

















19


ERF115 292

293














PCD (Fig 6) 307








20






319

320


21

Discussion 321












333












22














357











23



















385






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Parsed Citations

5













98












6













121












7




135

136


8

Results 137




























11

























12














process 198

199










13







tips 214

215

















14




15






238














252





16




precursor cells 259

260







17





18







275

















19


ERF115 292

293














PCD (Fig 6) 307








20






319

320


21

Discussion 321












333












22














357











23



















385






24















404








412


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Parsed Citations

Reviewer PDF

Parsed Citations

6













121












7




135

136


8

Results 137




























11

























12














process 198

199










13







tips 214

215

















14




15






238














252





16




precursor cells 259

260







17





18







275

















19


ERF115 292

293














PCD (Fig 6) 307








20






319

320


21

Discussion 321












333












22














357











23



















385






24















404








412


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428









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lt 005) 579

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Parsed Citations

Reviewer PDF

Parsed Citations

7




135

136


8

Results 137




























11

























12














process 198

199










13







tips 214

215

















14




15






238














252





16




precursor cells 259

260







17





18







275

















19


ERF115 292

293














PCD (Fig 6) 307








20






319

320


21

Discussion 321












333












22














357











23



















385






24















404








412


25
















428









26








443

















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28
















PRJNA380494 496









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543






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respectively) 560

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32


(bottom) 573

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lt 005) 579

580

581























































































































Parsed Citations

Reviewer PDF

Parsed Citations

8

Results 137




























11

























12














process 198

199










13







tips 214

215

















14




15






238














252





16




precursor cells 259

260







17





18







275

















19


ERF115 292

293














PCD (Fig 6) 307








20






319

320


21

Discussion 321












333












22














357











23



















385






24















404








412


25
















428









26








443

















27
























28
















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Figure Legends 534









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32


(bottom) 573

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lt 005) 579

580

581























































































































Parsed Citations

Reviewer PDF

Parsed Citations





11

























12














process 198

199










13







tips 214

215

















14




15






238














252





16




precursor cells 259

260







17





18







275

















19


ERF115 292

293














PCD (Fig 6) 307








20






319

320


21

Discussion 321












333












22














357











23



















385






24















404








412


25
















428









26








443

















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Figure Legends 534









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32


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lt 005) 579

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Parsed Citations

Reviewer PDF

Parsed Citations

12














process 198

199










13







tips 214

215

















14




15






238














252





16




precursor cells 259

260







17





18







275

















19


ERF115 292

293














PCD (Fig 6) 307








20






319

320


21

Discussion 321












333












22














357











23



















385






24















404








412


25
















428









26








443

















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28
















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543






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respectively) 560

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32


(bottom) 573

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lt 005) 579

580

581























































































































Parsed Citations

Reviewer PDF

Parsed Citations

13







tips 214

215

















14




15






238














252





16




precursor cells 259

260







17





18







275

















19


ERF115 292

293














PCD (Fig 6) 307








20






319

320


21

Discussion 321












333












22














357











23



















385






24















404








412


25
















428









26








443

















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28
















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respectively) 560

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32


(bottom) 573

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lt 005) 579

580

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Parsed Citations

Reviewer PDF

Parsed Citations

14




15






238














252





16




precursor cells 259

260







17





18







275

















19


ERF115 292

293














PCD (Fig 6) 307








20






319

320


21

Discussion 321












333












22














357











23



















385






24















404








412


25
















428









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Parsed Citations

Reviewer PDF

Parsed Citations

15






238














252





16




precursor cells 259

260







17





18







275

















19


ERF115 292

293














PCD (Fig 6) 307








20






319

320


21

Discussion 321












333












22














357











23



















385






24















404








412


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428









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Parsed Citations

Reviewer PDF

Parsed Citations

16




precursor cells 259

260







17





18







275

















19


ERF115 292

293














PCD (Fig 6) 307








20






319

320


21

Discussion 321












333












22














357











23



















385






24















404








412


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428









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Parsed Citations

Reviewer PDF

Parsed Citations

17





18







275

















19


ERF115 292

293














PCD (Fig 6) 307








20






319

320


21

Discussion 321












333












22














357











23



















385






24















404








412


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428









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580

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Parsed Citations

Reviewer PDF

Parsed Citations

18







275

















19


ERF115 292

293














PCD (Fig 6) 307








20






319

320


21

Discussion 321












333












22














357











23



















385






24















404








412


25
















428









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Parsed Citations

Reviewer PDF

Parsed Citations

19


ERF115 292

293














PCD (Fig 6) 307








20






319

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21

Discussion 321












333












22














357











23



















385






24















404








412


25
















428









26








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Parsed Citations

Reviewer PDF

Parsed Citations

20






319

320


21

Discussion 321












333












22














357











23



















385






24















404








412


25
















428









26








443

















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28
















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Parsed Citations

Reviewer PDF

Parsed Citations

21

Discussion 321












333












22














357











23



















385






24















404








412


25
















428









26








443

















27
























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Parsed Citations

Reviewer PDF

Parsed Citations

22














357











23



















385






24















404








412


25
















428









26








443

















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28
















PRJNA380494 496









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Parsed Citations

Reviewer PDF

Parsed Citations

23



















385






24















404








412


25
















428









26








443

















27
























28
















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580

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Parsed Citations

Reviewer PDF

Parsed Citations

24















404








412


25
















428









26








443

















27
























28
















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Parsed Citations

Reviewer PDF

Parsed Citations

25
















428









26








443

















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28
















PRJNA380494 496









29




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519



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Parsed Citations

Reviewer PDF

Parsed Citations

26








443

















27
























28
















PRJNA380494 496









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Reviewer PDF

Parsed Citations

27
























28
















PRJNA380494 496









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519



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Parsed Citations

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Parsed Citations

28
















PRJNA380494 496









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507












519



(AT1G25580) 522

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Parsed Citations

29




507












519



(AT1G25580) 522

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Figure Legends 534









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Parsed Citations

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Parsed Citations

30







533

Figure Legends 534









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549


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respectively) 560

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Parsed Citations

Reviewer PDF

Parsed Citations

31











respectively) 560

561




565








32


(bottom) 573

574





lt 005) 579

580

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Parsed Citations

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Parsed Citations

32


(bottom) 573

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lt 005) 579

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Parsed Citations






















































































































Parsed Citations

Reviewer PDF

Parsed Citations

1 Short title: 6 Affiliations - Plant Physiology · 5 Toal, Siobhan M. Brady, Anne B. Britt*(2) 6...

Documents

Transcript of 1 Short title: 6 Affiliations - Plant Physiology · 5 Toal, Siobhan M. Brady, Anne B. Britt*(2) 6...

1 Short title: 6 Affiliations - Plant Physiology · 5 Toal*, Siobhan M. Brady*, Anne B. Britt*(2) 6...

Documents

Transcript of 1 Short title: 6 Affiliations - Plant Physiology · 5 Toal*, Siobhan M. Brady*, Anne B. Britt*(2) 6...

1 Short title: 6 Affiliations - Plant Physiology · 5 Toal, Siobhan M. Brady, Anne B. Britt*(2) 6...

Transcript of 1 Short title: 6 Affiliations - Plant Physiology · 5 Toal, Siobhan M. Brady, Anne B. Britt*(2) 6...