1

Mitochondrial Defects Confer Tolerance against Cellulose Deficiency in Arabidopsis 1

2

Zhubing Hu,a,b,c Rudy Vanderhaeghen,a,b Toon Cools,a,b Yan Wang,e Inge De Clercq,a,b 3

Olivier Leroux,g Long Nguyen,h Katharina Belt,f A. Harvey Millar,f Dominique Audenaert,h 4

Pierre Hilson,a,b,d Ian Small,f Grégory Mouille,d Samantha Vernhettes,dFrank Van 5

Breusegem,a,b James Whelan,e Herman Höfte,d and Lieven De Veyldera,b,1 6 7 aDepartment of Plant Systems Biology, VIB, B-9052 Gent, Belgium 8 bDepartment of Plant Biotechnology and Bioinformatics, Ghent University, B-9052 Gent, 9

Belgium 10 cCollege of Life Sciences, Nanjing Agricultural University, 210095 Nanjing, People’s 11

Republic of China 12 dInstitut Jean-Pierre Bourgin, INRA, Centre National pour la Recherche Scientifique, 13

AgroParisTech, Université Paris-Saclay, RD10, 78026 Versailles Cedex France 14 eDepartment of Botany, ARC Centre of Excellence in Plant Energy Biology, School of Life 15

Science, La Trobe University, Bundoora 3086, Victoria, Australia 16 fAustralian Research Council Centre of Excellence in Plant Energy Biology, University of 17

Western Australia, Crawley 6009, Australia 18 gDepartment of Biology, Ghent University, K.L. Ledeganckstraat 35, Ghent B-9000, Belgium 19 hCompound Screening Facility, VIB, B-9052 Gent, Belgium 20

21

ORCID IDs: 0000-0002-1258-5531 (T.C); 0000-0001-8125-1239 (I.D.C.); 0000-0001-5300-22

1216 (I.S.); 0000-0003-4617-1908 (P.H.); 0000-0002-3147-0860 (F.V.B); 0000-0003-1150-23

4426 (L.D.V.); 0000-0001-5754-025X (J.W); 24

25

Running title: Mitochondria mediate cell wall integrity 26

27

Corresponding author: 28

Lieven De Veylder 29

Department of Plant Systems Biology 30

VIB-Ghent University 31

Technologiepark 927, B-9052 Gent (Belgium). 32

Tel.: +32 9 3313800; Fax: +32 9 3313809; E-mail: lieven.deveylder@psb.vib-ugent.be 33

Footnotes 34

2

1Address correspondence to lieven.deveylder@psb.vib-ugent.be. 35

The author responsible for distribution of materials integral to the findings presented in this 36

article in accordance with the policy described in the Instructions for Authors 37

(www.plantcell.org) is: Lieven De Veylder (lieven.deveylder@psb.vib-ugent.be). 38

39

3

ABSTRACT 40

Because the plant cell wall provides the first line of defence against biotic and abiotic 41

assaults, its functional integrity needs to be maintained under stress conditions. Through 42

a phenotype-based compound screening approach we identified a novel cellulose 43

synthase inhibitor, designated C17. C17 administration depletes cellulose synthase 44

complexes (CSCs) from the plasma membrane, resulting in anisotropic cell elongation 45

and a weak cell wall. Surprisingly, next to mutations in cellulose synthase 1 (CESA1) and 46

cellulose synthase 3 (CESA3), a forward genetic screen identified two independent 47

defective pentatricopeptide repeat (PPR)-like proteins (Cell Wall Maintainer 1, (CWM1) 48

and 2 (CWM2)) to confer tolerance to C17. Functional analysis revealed that mutations 49

in these PPR genes resulted in defective cytochrome c maturation and activation of 50

mitochondrial retrograde signalling, as evidenced by the induction of an alternative 51

oxidase. These mitochondrial perturbations increased tolerance to cell wall damage 52

induced by cellulose deficiency. Likewise, administration of Antimycin A, a well-53

characterized inhibitor of mitochondrial complex III, and constitutive activation of 54

mitochondrial retrograde signalling resulted in tolerance towards C17. The C17 55

tolerance of cwm2 was partially lost upon depletion of the mitochondrial retrograde 56

regulator ANAC017, demonstrating that ANAC017 links mitochondrial dysfunction 57

with the cell wall. In view of mitochondria being a major target of a variety of stresses, 58

our data indicate that plant cells might modulate mitochondrial activity to maintain a 59

functional cell wall when subjected to stresses. 60

61

4

INTRODUCTION 62

Cellulose is a main component of the plant cell wall, consisting of a linear chain of several 63

hundred to many thousands of β-1,4 linked D-glucose units (McFarlane et al., 2014). Its 64

synthesis is achieved through a plasma membrane (PM)-localized protein complex referred to 65

as the cellulose synthase complex (CSC) that has a hexameric rosette-like structure (Endler 66

and Persson, 2011). Each of the six lobes is thought to contain three distinct cellulose 67

synthase catalytic subunits (CESAs) corresponding to CESA1, CESA3 and CESA6 (or 68

CESA6-like proteins CESA2, CESA5 and CESA9) in the primary cell wall (Desprez et al., 69

2007; Persson et al., 2007); (Vandavasi et al., 2016). 70

The activity of the CSC is modulated by abiotic and biotic stresses. For example, when 71

exposed to osmotic stress, plants downregulate their cellulose production through the 72

depletion of CSCs from the plasma membrane (Gutierrez et al., 2009; Lei et al., 2015). A 73

similar depletion can be triggered by small molecules produced by pathogens, such as 74

thaxtomin A, a potent inhibitor of cellulose biosynthesis produced by the plant pathogen 75

Streptomyces scabies responsible for the scab disease (Crowell et al., 2009). Recently, a novel 76

CSC inhibitor acetobixan was isolated from small molecule secretions derived from a library 77

of switchgrass endophytes (Xia et al., 2014). 78

Because the cell wall is a key feature of plant cells, dedicated systems have evolved to 79

monitor its integrity and to trigger changes in its composition and structure through tightly 80

controlled enzymatic modifications and shifts in cellular metabolism (Hamann, 2015). Cell 81

wall damage induces a wide range of responses, including ectopic lignin deposition, 82

activation of jasmonate and ethylene signalling pathways, and upregulation of stress response 83

genes (Caño-Delgado et al., 2000; Ellis and Turner, 2001; Ellis et al., 2002; Caño-Delgado et 84

al., 2003). Several proteins have been identified to control these responses (Wolf et al., 2012). 85

Among these, THESEUS1 (THE1) is one of the best-studied components, belonging to the 86

family of the Catharanthus roseus receptor-like kinases. THE1 was originally identified in a 87

screen for the suppression of the elongation defect in the cellulose-deficient mutant 88

cesa6/procuste1 (prc1). Depletion of THE1 partially restores growth inhibition and attenuates 89

ectopic lignification of cellulose-deficient mutants, but fails to rescue the cellulose deficiency 90

(Hématy et al., 2007). 91

In addition to the cell wall, mitochondria have been considered to be the target of a variety 92

of stresses (Bartoli et al., 2004; Giraud et al., 2008). Generally, stresses can trigger the 93

accumulation of reactive oxygen species (ROS), which in turn result in oxidative damage to 94

5

mitochondria (Fujita et al., 2006; Gechev et al., 2006). Apart from being a target of ROS, 95

mitochondria are a major source of cellular ROS (Rhoads et al., 2006). Mitochondria possess 96

a large number of cellular enzymatic systems that catalyse the oxidation of various substrates 97

and generate the reducing equivalents to reduce the pyridine and flavin nucleotides NAD and 98

FAD. Reduced NAD and FAD in turn are oxidized by coenzyme Q (CoQ) in reactions 99

catalysed by several enzyme complexes located in the inner membrane of mitochondria. 100

Ultimately, the flux of electrons from substrates through various redox carriers and centres is 101

terminated in a four-electron reduction of molecular oxygen to water, catalysed by the 102

cytochrome c oxidase (Starkov, 2008). Stress-induced inhibition of the various mitochondrial 103

electron transport protein complexes might result in an increase in the non-enzymatic single-104

electron reduction of oxygen, converting it into superoxide, a progenitor ROS (Starkov, 2008; 105

Millar et al., 2011; Huang et al., 2016). Although many mitochondrial mutants exhibit 106

increased ROS levels (Zsigmond et al., 2008; Liu et al., 2010; He et al., 2012; Yang et al., 107

2014; Zhu et al., 2014), a recent study reported that inhibiting mitochondrial activity might 108

play a protective role to prevent the production of mitochondrial ROS and diminish the ROS-109

induced damage (Wu et al., 2015). Moreover, perturbing mitochondrial functions activates 110

signalling cascades from this organelle to the nucleus, resulting in the modulation of gene 111

expression (Rhoads and Subbaiah, 2007; Ng et al., 2014; Huang et al., 2016). In plants, 112

several signalling components have been identified for this process, which include the cyclin-113

dependent kinase E1 (CDKE1), as well as the transcription factors ABSCISIC ACID 114

INSENSITIVE 4 (ABI4), WRKY40, NO APICAL MERISTEM/ARABIDOPSIS 115

TRANSCRIPTION FACTOR/CUP-SHAPED COTYLEDON013 (ANAC013), and 116

ANAC017 (Giraud et al., 2009; De Clercq et al., 2013; Ng et al., 2013b; Ng et al., 2013a; Van 117

Aken et al., 2013). The depletion of either ANAC013 or ANAC017 affect mitochondrial 118

retrograde signalling, resulting in plants being hypersensitive to abiotic stresses (De Clercq et 119

al., 2013; Ng et al., 2013a), which highlights the importance of mitochondria in stress 120

adaptation. Comparative analysis shows that among related NACs, ANAC017 is almost solely 121

responsible for transcript induction of mitochondrial retrograde marker genes after chemical 122

inhibition of organelle function (Van Aken et al., 2016). 123

Despite the crucial role of the plant mitochondria and the cell wall in stress response, it is 124

unclear whether these two compartments are functionally linked. Here, we report a novel CSC 125

inhibitor named C17 that reduces cellulose production through perturbation of CSC activity. 126

Surprisingly, C17-induced growth defects can be suppressed by perturbation of mitochondrial 127

6

activity and by activation of mitochondrial retrograde signalling, indicating that modulation of 128

mitochondrial activity might be required for the maintenance of functional cell walls. 129

130

RESULTS 131

C17 Interferes with Cytokinesis in the Process of Mitotic Division 132

Because of their sessile lifestyle, plants cannot escape unfavourable growth conditions, which 133

make them vulnerable to environmental stress conditions that interfere with a series of 134

physiological processes, ultimately resulting in a cell cycle exit and growth inhibition. 135

Compounds that interfere with cell division processes can be identified by assessment of 136

intracellular DNA accumulation. To screen for novel cell division interfering compounds, a 137

high-throughput chemical screen was performed using an Arabidopsis cell suspension line 138

producing a translational fusion between the Arabidopsis Histone 2B and Yellow Fluorescent 139

Protein genes (H2B-YFP). Because H2B-YFP associates with chromatin and is fluorescently 140

marked, DNA accumulation can be indirectly measured by enhanced YFP fluorescence. The 141

H2B-YFP cells were treated with a chemical library of 12,000 organic molecules 142

(DIVERSetTM, ChemBridge Corporation, San Diego, California, USA). Three compounds 143

were found to induce polyploidy within 72 h of treatment, of which the synthetic molecule 144

C17 (5-(4-chlorophenyl)-7-(2-methoxyphenyl)-1,5,6,7-tetrahydro-[1,2,4]triazolo[1,5-a] 145

pyrimidine; ChemBridge, Catalogue #: 7693622) was the most effective (Figure 1A). C17-146

treated cells showed larger nuclei and higher YFP fluorescence (Figure 1B). Through time-147

lapse imaging, C17 was found to inhibit cytokinesis without affecting mitosis, thus leading to 148

endomitosis (Figure 1C, Supplemental Video 1 and 2). 149

150

Mutations in CESA1 and CESA3 Confer C17 Tolerance 151

In wild-type plants (Col-0), C17 administration resulted in a dose-dependent inhibition of 152

cotyledon expansion and root growth, accompanied by the radial swelling of the root tip, with 153

an IC50 < 0.1 µM (Figure 2A and 2B). To gain insight into the growth inhibitory activity of 154

C17, an ethylmethanesulfonate (EMS)-based genetic screen was performed to identify 155

mutants that display tolerance to an extreme growth-inhibitory dose of C17 (2 µM), resulting 156

in a total of 22 C17-tolerant mutants. All 22 mutants, except for 9R and 18A1 having a slight 157

growth penalty, were phenotypically indistinguishable from wild-type plants in the absence of 158

7

C17 (Figure 2C), whereas the C17 inhibitory activity was attenuated in these C17-tolerant 159

mutants which, in the presence of the compound, had longer roots and bigger cotyledons 160

compared with wild-type seedlings (Figure 2D). 161

Based on C17 sensitivity, the segregation ratio of F2 progenies indicated that 15 mutants 162

displayed semi-dominant phenotypes (1:2:1 ratio, sensitive:intermediate tolerant:tolerant), 163

whereas seven mutants exhibited a recessive phenotype (3:1 ratio, sensitive:tolerant) (Table 164

1), thus indicating that C17 tolerance resulted from single-gene mutations. All mutants were 165

crossed with the Ler-0 ecotype to generate mapping populations. Linkage analysis with 24 166

simple sequence length polymorphism (SSLP) markers divided the 22 mutants into two 167

groups labelled with the name of the closest corresponding markers, CH4-14494 and CH5-168

512 respectively (Supplemental Figure 1). Fine mapping and genome sequencing of the 7L 169

and 2C mutant alleles identified the causal nucleotide mutation in the CELLULOSE 170

SYNTHASE A1 (CESA1) and CELLULOSE SYNTHASE A3 (CESA3) loci, respectively (Figure 171

3A and 3B). The CESA1 and CESA3 loci of the remaining C17-tolerant mutants were 172

sequenced, revealing that all identified C17-tolerant mutants carried a single-nucleotide 173

missense change at either CESA1 or CESA3, all resulting in an amino acid change (Table 1). 174

Collectively, this mutant series consists of ten mutant alleles of CESA1 and two of CESA3 175

(Table 1). Protein sequence analysis showed that most mutated amino acids clustered to the 176

transmembrane (TM) regions of the CESA proteins (Figure 3C). Furthermore, amino acid 177

alignment of CESA1/CESA3 homologues from seven species revealed that ten of these 12 178

mutated amino acids are invariant (Supplemental Figure 2). 179

180

C17 Inhibits Cellulose Biosynthesis and Acts via Clearance of CSCs from the Plasma 181

Membrane 182

C17 inhibits hypocotyl elongation (Figure 4A and 4B), correlated with a decrease in cellulose 183

content, as measured by the amount of glucose produced through hydrolysis of cellulose 184

(Figure 4C). Consistently, mutations encoding C17 tolerance revert the inhibition of 185

hypocotyl elongation (Figure 4A and 4B). The effect of C17 on CESA activity was addressed 186

through live cell imaging using CESA3-GFP reporter plants. Compared with untreated 187

hypocotyl epidermal cells, administration of C17 resulted in a significant reduction of the 188

CESA3-GFP signal associated with the plasma membrane (Figure 4D). These results 189

demonstrate that C17’s impact on cellulose is caused by a removal of the CESA complexes 190

from the plasma membrane. Cortical microtubules guide the movement of the CESA 191

8

complexes in the plasma membrane (Crowell et al., 2009; Gutierrez et al., 2009). C17 192

application did not affect microtubule organization, demonstrating that the observed CESA3-193

GFP depletion was not due to microtubule depolymerization (Supplemental Figure 3). 194

195

C17-Induced Depletion of Membrane CESAs Results in a Weaker Cell Wall 196

Similar to its effect on the hypocotyl, C17 treatment resulted in the depletion of the CESA 197

complex from the plasma membrane of wild-type root cells with a dramatic drop after 10 to 198

15 min of C17 application (Supplemental Figure 4). Because the cellulose synthesized by the 199

CESA1/CESA3 complex is a primary cell wall component, it was expected that C17-treated 200

plants would display a weaker cell wall, which can be visualized by the uptake of propidium 201

iodide (PI) following the application of a gentle pressure on the root (see Materials and 202

Methods). Indeed, cell wall weakening was observed in the root elongation zone within 2 h 203

after applying C17, which increased over time (Figure 5A, left panel) and correlated with 204

growth inhibition (Figure 5C). Likewise, a weaker cell wall could be visualized in the root 205

elongation zone of je5 plants that are mutant in CESA3 (Figure 5B), indicating that the 206

phenotypes observed upon C17 treatment correlate with cellulose deficiency. No PI-positive 207

cells and growth inhibition were observed in C17-tolerant mutants (Figure 5A, right panel and 208

5C). 209

210

Mutations in PPR-Like Genes Counteract the Growth Inhibition Induced by Cellulose 211

Deficiency 212

To isolate putative components responding to CESA deficiency, a second mutagenesis screen 213

was performed at a low dose of C17 (200 nM) using T-DNA insertion lines. At this moderate 214

concentration of C17 root growth is inhibited instantly upon transfer to C17-containing 215

medium (Figure 5C). Two insertion lines (SALK_017325C and SALK_020569C, hereafter 216

referred to as cell wall maintainer1-1 (cwm1-1) and cwm2-1, respectively) exhibited 217

significant suppression of the growth inhibition in the presence of C17 (Figure 6A and 6B). 218

The cwm1-1 line has a T-DNA insertion in the AT1G17630 locus, cwm2-1 in AT5G44570. 219

Suppression of C17 sensitivity by AT1G17630 deficiency was confirmed with two 220

independent T-DNA insertion lines (SALK_124160, cwm1-2 and SALK_078133, cwm1-3) of 221

this locus. The T-DNA inserts in these three lines are positioned in the region of AT1G17630 222

encoding a predicted pentatricopeptide repeat (PPR)-like protein (Supplemental Figure 5A). 223

9

Although the annotated cwm2-1 mutant (SALK_020569C) is a knockout of AT5G44570, 224

an independent T-DNA insertion line (SAIL_699_C11) could not suppress the C17-induced 225

growth inhibition (Supplemental Figure 5B). Furthermore, C17 tolerance in cwm2-1 was 226

unlinked to the T-DNA insert in the AT5G44570 locus, confirming AT5G44570 is not the 227

CWM2 gene. To isolate the CWM2 gene, we crossed cwm2-1 with the Ler ecotype and carried 228

out the positional cloning of the causal mutation. The CWM2 gene was mapped into a 171.8-229

kb region, between SSLP markers CH1_11687 and CH1_11859 (Supplemental Figure 5C). 230

PCR analysis of T-DNA flanking sequences identified a second T-DNA insert positioned in 231

the coding region of the AT1G32415 locus that also encodes a PPR-like superfamily protein. 232

In addition to cwm2-1, the SALK_027874 line (cwm2-2) that harbours an independent T-233

DNA insert resulting in the depletion of the full-length mRNA transcribed from the 234

AT1G32415 locus (Supplemental Figure 5C) also exhibits C17 tolerance (Figure 6A and 6B), 235

thus demonstrating that AT1G32415 encodes the CWM2 protein. In addition, both cwm1 and 236

cwm2 mutants displayed an enhanced tolerance to two other cellulose synthase inhibitors, 237

isoxaben and indaziflam(Desprez et al., 2002; Brabham et al., 2014) (Supplemental Figure 6). 238

239

Both cwm1 and cwm2 Mutations Affect Mitochondrial Complexes 240

Protein domain analysis indicated that both CWM1 and CWM2 proteins contain an amino-241

terminal mitochondrial targeting peptide, strongly suggesting that CWM1 and CWM2 are 242

localized in the mitochondria. Since PPR proteins have been found to affect the maturation, 243

stability or expression of transcripts encoded in the mitochondrial genome (Barkan and Small, 244

2014), the mitochondrial transcriptome of the cwm1 and cwm2 mutants was sequenced. In 245

cwm1 transcripts, editing defects were found in the transcripts of cytochrome c maturation 246

protein B (ccmB), cytochrome c maturation protein C (ccmC), and NADH dehydrogenase 247

subunit 5 (nad5) sequences (Figure 7A), while an editing defect in the ccmC sequence was 248

identified in the cwm2 transcript (Figure 7B). nad5 encodes a subunit of NADH 249

dehydrogenase in mitochondrial complex I, whereas both ccmB and ccmC are required for the 250

maturation of cytochrome c (Kranz et al., 2009; Millar et al., 2011). Consistently, a dramatic 251

decrease in cytochrome c content was observed in all cwm mutants, whereas the level of the 252

ubiquinone oxidoreductase Fe-S protein4 (NDUFS4, subunit of complex I) was affected only 253

in the cwm1 mutants (Figure 8A and 8B). Three proteins were additionally found to display 254

altered abundances in all cwm mutants, with cytochrome oxidase subunit II (COXII, subunit 255

of complex IV) and alternative oxidase (AOX) being detected at higher levels, and the Rieske 256

10

iron–sulfur protein (RISP, subunit of complex III) being reduced (Figure 8A and 8B). No 257

significant change in protein abundance was observed for the beta subunit of ATP synthase 258

(ATPβ, subunit of complex V) and three tested mitochondrial import inner membrane 259

translocase subunits (Tim9, Tim17 and Tim23) (Figure 8A and 8B). 260

To determine the abundance of assembled respiratory complexes, total membrane proteins 261

were resolved by blue native polyacrylamide gel electrophoresis, and specific complexes were 262

detected by probing immunoblots with antibodies against NDUFS4 (complex I), RISP 263

(complex III), COXII (complex IV) and ATPβ (complex V) (Figure 8C). The results showed 264

that assembled complex V accumulates to normal levels in all cwm mutants, but assembled 265

complex IV accumulated to higher levels and assembled dimeric complex III to lower levels 266

(Figure 8C). A clear accumulation of supercomplex I-III2 composed of complex I and a dimer 267

of complex III and supercomplex I2III4, a dimer of I-III2 was observed in cwm2 mutants 268

(Figure 8C). Consistent with the mis-edited NAD5 transcript in the cwm1 transcriptome, 269

complex I was less abundant in these mutants (Figure 8C). Complexes staining further 270

confirmed the changes of assembled complex I and complex IV (Supplemental Figure 7). 271

As both cwm1 and cwm2 mutations resulted in the changes in the abundance of assembled 272

respiratory complexes, we measured their respiration rates. Compared with wild-type 273

seedlings, no significant difference was detected in all four cwm mutants under normal growth 274

condition. However, although all seedlings showed a decreased respiration in presence of 275

potassium cyanide (KCN) that inhibit cytochrome pathway, the cwm mutants displayed a 276

higher respiration than wild type, indicating increased AOX activity in all the cwm mutants 277

(Supplemental Figure 8). 278

To confirm that mitochondrial defects lay at the origin of a signalling cascade that confer 279

C17-tolerance, we applied antimycin A (AA), a well-characterized inhibitor of mitochondrial 280

complex III, and rotenone (RO), a complex I inhibitor, to wild-type plants. Similar to the 281

cwm1 and cwm2 mutants, AA suppressed the C17-induced growth inhibition (Figure 9A and 282

9B) and reversed the brittle cell wall phenotype (Figure 9C). Contrastingly, RO did not confer 283

C17 tolerance (Figure 9A and 9B). Consistently, several mutants (ndufs4, bir6-2 and otp439) 284

with defective complex I exhibited the same C17 sensitivity as wild type plants (Supplemental 285

Figure 9) (de Longevialle et al., 2007; Meyer et al., 2009; Koprivova et al., 2010). 286

Although C17 inhibits cellulose production by depletion of CSCs from the plasma 287

membrane, AA administration did not reverse this phenotype (Figure 9D to 9G and 288

Supplemental Figure 10), indicating that the inhibition of mitochondrial activity did not 289

restore CSC activity itself. Reversely, C17 had no significant effect on respiration rate, even 290

11

at a concentration as high as 8 µM, ruling out the possibility that C17 directly acts on the 291

cytochrome pathway of respiration (Supplemental Figure 11). 292

293

Both cwm1 and cwm2 Mutations Enhance the Tolerance of je5 against Osmotic Stress 294

The cell wall is anticipated to help plants to cope with environmental stimuli, such as osmotic 295

stresses (Zhu et al., 2010; Tenhaken, 2015). Correspondingly, C17 treated plants and the je5 296

mutant show a severe root growth inhibition in response to mannitol (Figure 10A and 10B). 297

Strikingly, whereas no significant difference in tolerance was observed between wild-type 298

plants and cwm single mutants, both cwm1 and cwm2 mutations rescued the osmotic stress 299

phenotype of the je5 mutant (Figure 10B and 10C), indicating that both cwm mutations confer 300

a functional cell wall to the je5 mutant for growth in presence of mannitol. 301

302 The C17 Tolerance Triggered by Dysfunctional Mitochondria Partially Depends on 303

ANAC017 304

Recently, the ANAC017 transcription factor was characterized as a key regulator in the 305

organelle signalling that links dysfunctional mitochondria and primary stress responses (Ng et 306

al., 2013a). To test whether ANAC017-dependent signalling might contribute to the tolerance 307

towards C17, ANAC017 overexpression plants were generated and tested for C17 sensitivity. 308

Two independent ANAC017OE lines exhibited higher C17 tolerance than wild-type plants 309

(Figure 11A and 11B). Reversely, the loss of ANAC017 activity partially compromised the 310

cwm2-1 triggered C17 tolerance, as revealed by the shorter roots and more isotropic shaped 311

cells of anac017-1 cwm2-1 double mutants compared to cwm2-1 single mutants, whereas no 312

difference in tolerance was observed between the wild type and ANAC017 knockout mutants 313

(Figure 11C to 11I). These data illustrate that ANAC017 is a signalling component linking 314

mitochondria to the cell wall. 315

316

DISCUSSION 317

Here, we report a novel CSC inhibitor (C17) severely impacting plant growth and 318

development at the nanomolar level. C17-triggered growth inhibition can be overcome by a 319

mutation in CESA1 or CESA3 without any growth penalty. Although being structural different 320

from known CSC-inhibiting compounds, including isoxaben, thaxtomin and quinoxyphen, 321

C17 also triggers a rapid loss of CSCs from the plasma membrane and the inhibition of 322

12

cellulose accumulation. The resulting cell wall is weakened as shown by its rupturing when 323

applying slight pressure on the surface of the organ. Mutations in CESA1 or CESA3 loose 324

this cell wall weakening, suggesting a causal link between C17, reduced CESA activity and 325

the weakening of the cell wall. A role as inhibitor of primary cell wall biosynthesis likely 326

explains the ploidy-inducing effect of C17 in cell cultures, which arises because of impaired 327

cytokinesis. During cytokinesis, a new cross wall is rapidly laid down, which requires 328

cellulose. It has been shown that cellulose and CESA1, CESA3 and CESA6 accumulate in the 329

developing cell plate that forms during cytokinesis (Miart et al., 2014). Correspondingly, the 330

cesa1rsw1-20 mutation results in incomplete cell plates (Beeckman et al., 2002). 331

The mechanism of plasma membrane depletion of CSCs triggered by C17 remains 332

unclear. This depletion was not an indirect consequence of cortical microtubule 333

depolymerisation. All twelve C17-tolerant EMS-induced mutant alleles encode an amino-acid 334

change in either CESA1 or CESA3, always located in or near the transmembrane spanning 335

region. Although the mutated amino acids are distributed over different transmembrane 336

domains, it might be that all mutated amino acids cluster together. The amino-acid changes in 337

C17- tolerant mutants might either directly or, indirectly alter the interaction between C17 and 338

CSCs. 339

In addition to mutant cesa1 and cesa3 alleles, two mutant ppr-like genes (cwm1 and cwm2) 340

were found to confer C17 tolerance. In recent years, a number of mitochondrial PPR-like 341

proteins have been characterized and found to participate in virtually all post-transcriptional 342

processes such as RNA editing, RNA splicing, and transcript processing (Schmitz-343

Linneweber and Small, 2008). Some of them are involved in RNA editing of single sites 344

(Takenaka, 2010; Takenaka et al., 2010), others control multiple sites (Zehrmann et al., 2009; 345

Bentolila et al., 2010; Sung et al., 2010; Verbitskiy et al., 2010). Both CWM1 and CWM2 346

play a role in mitochondrial RNA editing: CWM1 was found to control three specific editing 347

sites (ccmB, ccmC and nad5), whereas CWM2 controls a single site (ccmC). The cwm1 and 348

cwm2 mutants have several common mitochondrial characteristics: reduced cytochrome c 349

content, mutated ccmC, and altered assembly of complex III and increased abundance of 350

complex IV, demonstrating that CWM1 or CWM2 deficiency causes dysfunctional 351

mitochondria. This is further supported by an increased abundance of AOX (Vanlerberghe, 352

2013). The most likely explanation for the defective complex III might be the altered 353

cytochrome c maturation machinery, which is needed to attach the heme to both the 354

cytochrome c1 in complex III, as well to the soluble cytochrome c (Mavridou et al., 2013). 355

The increased abundance of complex IV is interesting because the same changes do not occur 356

13

in a partial cytochrome c loss-of-function mutant (Welchen et al., 2012). These mutants 357

display a reduction in cytochrome c levels (Welchen et al., 2012). In our case, both CWM1 358

and CWM2 encode editing proteins required for the maturation of cytochrome c, rather than 359

the components in mitochondrial complexes. This may indicate that the signals resulting from 360

a lack of a structural gene differ from those preventing assembly of a functional complex. 361

Likewise, mitochondrial mutants with a high AOX content display different changes in the 362

mitochondrial complex compared with those of cwm1 and cwm2 mutants (Francs-Small et al., 363

2012). 364

C17 tolerance of both cwm1 and cwm2 mutants may be attributed to the decreased activity 365

of mitochondrial complex III. In support of this hypothesis, administration of the 366

mitochondrial complex III inhibitor AA partially reverses the C17 sensitivity phenotype 367

(Figure 9A). AA blocks the Qi site and loss of the heme on Cyt c1 would block the final 368

electron transfer to cytochrome c. Both would leave the Qo site active, which is linked to 369

ROS signalling (Bleier and Dröse, 2013). Likely, there is some rather specific ROS 370

production at the blocked complex III, resulting in retrograde signaling that leads to C17 371

tolerance. Contrastingly, the complex I mutants (ndufs4, bir6-2 and otp439) and the chemical 372

inhibitor RO could not confer C17 tolerance to plants, suggesting that a defective 373

mitochondrial complex I in cwm1 mutants is not linked to C17 tolerance. These observations 374

exclude the possibility that the alleviated deleterious effects in cwm mutants are caused by 375

reduced growth as a result of reduced respiration with dysfunctional mitochondria. Although 376

we cannot absolutely rule out the possibility that C17 acts on mitochondria, it seems very 377

unlikely because high dosage of C17 did not cause inhibition of the cytochrome pathway of 378

respiration (Supplemental Figure 11). Additionally, the increased tolerance of cwm1 and 379

cwm2 towards two other cellulose synthase inhibitors emphasizes cwm1 and cwm2 mutations 380

counteract the growth inhibition induced by cellulose deficiency rather than other effects 381

triggered by C17. 382

Perturbing mitochondria alters nuclear gene expression by organelle signalling regulation 383

(Rhoads and Subbaiah, 2007; Ng et al., 2014). Organelle signalling is active in the cwm1 and 384

cwm2 mutants, as marked by AOX accumulation, and might mediate a crosstalk between the 385

mitochondria and the cell wall. This hypothesis is supported by the observation that ANAC017 386

overexpression confers C17 tolerance, whereas ANAC017 depletion result in a partial loss of 387

the cwm2-1 triggered C17 tolerance phenotype. ANAC017 is the core cellular component in 388

mitochondrial organelle signalling, particularly for retrograde signalling, accounting for more 389

than 85% of H2O2-mediated primary stress responses in plants (Ng et al., 2013a). Because 390

14

ANAC017 is latent in the endoplasmic reticulum (ER) and activated by mitochondrial 391

dysfunction (Ng et al., 2013a), C17 tolerance upon inhibition of mitochondrial activity might 392

find its origin in the ER, because all the cell wall-synthesizing enzymes are transported via the 393

ER. ANAC017 deficiency only partially abolished C17 tolerance triggered by cwm2-1 394

mutation. Possibly, ANAC017 functions redundantly with another ER-bound NAC 395

transcription factor, such as ANAC013 (De Clercq et al., 2013) or ANAC017-independent 396

organelle signalling cascades. Indeed, at least two other stress responsive systems in the ER 397

are known, including the activation of bZIP transcription factors by the S1P and S2P 398

proteases and the IRE1 and IRE2 splicing systems (Deng et al., 2013; Howell, 2013; 399

Srivastava et al., 2013). Moreover, overactivation of these systems do not require functional 400

ANAC017, and vice versa, they were not required for activation (Ng et al., 2013a). 401

Our data suggest that the inhibition of mitochondria contributes to the maintenance of a 402

functional cell wall under osmotic stress conditions. In agreement, CWM1 or CWM2 loss of 403

function in cellulose-deficient plants increased the tolerance to osmotic stresses, indicating 404

that the cell wall in these plants copes better with stress. Enhanced osmotic tolerance was only 405

observed in a cellulose-deficient background, indicating that the connection between 406

mitochondrial function and cell wall is masked in wild-type plants. Alternatively, regarding 407

the fact that osmotic stress naturally results in decreased cellulose production by depletion of 408

membrane-associated CESAs (Crowell et al., 2009), these changes might be dependent on the 409

cell wall status. Similar repression of cellulose production was observed in several different 410

species including Arabidopsis, tobacco suspension cells, grape leaves and wheat roots (Iraki 411

et al., 1989; Sweet et al., 1990; Piro et al., 2003; Bray, 2004; Crowell et al., 2009). 412

Taken together, our results suggest that perturbing mitochondrial activity results in the 413

attenuation of the growth inhibition that is caused by CESA depletion from the plasma 414

membrane, likely through a change in cell wall composition. Moreover, consistent with the 415

inhibited mitochondrial activity and their importance in the adaption to osmotic stresses 416

(Skirycz et al., 2010; Vanderauwera et al., 2012), our data point to a possible mechanism 417

underlying adaptation to osmotic stress, in which the modulation of mitochondrial activity and 418

subsequent retrograde signaling controls the maintenance of cell wall integrity. 419

420

METHODS 421

Plant Materials and Growth Conditions 422

15

Arabidopsis thaliana plants were grown under long-day conditions (16 h of light/8 h of 423

darkness) at 22°C on half-strength Murashige and Skoog (MS) germination medium 424

(Murashige and Skoog, 1962). The cwm1-1 (SALK_017325C), cwm1-2 (SALK_124160), 425

cwm1-3 (SALK_078133), cwm2-1 (SALK_020569C), cwm2-2 (SALK_027874) and 426

SAIL_699_C11 were acquired from the ABRC. The otp439, bir6-2, ndufs4, je5, and 427

anac017-1 mutants, and the GFP-CESA3 line in the cesa3je5 background have been described 428

previously (de Longevialle et al., 2007; Desprez et al., 2007; Meyer et al., 2009; Koprivova et 429

al., 2010; Ng et al., 2013a). 430

431

Chemical Treatments 432

Antimycin A (AA), and rotenone (RO) were purchased from Sigma-Aldrich and applied from 433

50 mM stock solutions in DMSO to the final concentration described in the text. Mock 434

treatments of 0.1 % (v/v) DMSO were used as a control. For the treatments of chemical 435

compounds, plants were grown on the control medium for three days and then transferred to 436

medium without or with the indicated drugs for two days. The 2-day elongation of seedling 437

roots after treatment was measured. 438

439 Generation of Transgenic Arabidopsis Plants 440

Overexpressing plants were generated by cloning the open reading frame (ORF) of ANAC017 441

into pK7WG2D (Karimi et al., 2002). Constructs were transformed into Arabidopsis Col-0 by 442

Agrobacterium tumefaciens-mediated floral dipping (Clough and Bent, 1998). 443

444

Chemical Screen 445

The PSB-L_H2B-YFP cell line was obtained through co-cultivation of the Arabidopsis 446

thaliana PSB-L cell suspension culture (Landsberg erecta) (May and Leaver, 1993) with an 447

A. tumefaciens strain carrying a binary T-DNA vector containing a transgene encoding a 448

translational fusion between the Arabidopsis Histone 2B (AT5G22880) and the Yellow 449

Fluorescent Protein (H2B-YFP) (Boisnard-Lorig et al., 2001). Protoplasts were isolated from 450

100 mL 3-day-old Arabidopsis PSB-L_H2B-YFP cell suspension culture expressing the H2B-451

YFP transgene. Cells were harvested through sedimentation in 2x50 mL tubes (Corning) and 452

removing the supernatants. After cell wall digestion with 100 mL cellulase (15 mg mL-1; 453

Yakult ‘Onozuka’ R10) in 0.4 M mannitol, 5 mM MES pH 5.7 for 3 to 4 hours at 250C, 50 454

rpm in a large (142/20 mm) vented Petri dish (Greiner), isolated cells were transferred to 455

16

2x50mL tubes (Corning) and washed with and resuspended in 25 mL MSMO containing 0.35 456

M mannitol, 0.5 mg L-1 NAA and 0.05 mg L-1 kinetin using centrifugation at 850 rpm. 457

Protoplasts were then filtered through a 40-µm mesh and incubated overnight in the dark at 458

room temperature for partial cell wall regeneration in MSMO containing 0.35 M mannitol, 0.5 459

mg L-1 NAA and 0.05 mg L-1 kinetin. Dead cells were removed by sucrose gradient (0.5 M) 460

decantation at 100 g during 10 min. Subsequently, the protoplasts were washed in 2x40 mL 461

and resuspended in 2x2.5-5 mL MSMO containing 0.35 M mannitol, 0.5 mg L-1 NAA and 462

0.05 mg L-1 kinetin in 2x50 mL tubes. Cell density was estimated with a cell counter 463

(CountessTM Automated Cell Counter, Invitrogen), and subsequently diluted to obtain a 464

standard density of 105 cells mL-1. Diluted cells were then seeded in a NuncTM 96-well CC2 465

Coverglass Bottom Plate (Thermo Scientific) at 100 µL per well using a Biomek® 2000 robot 466

(Beckman Coulter). Subsequently, compounds of the DIVERSet library (12,000 compounds) 467

were added with a Te-MO robot (Tecan) to a final concentration of 50 µM (1 % (v/v) 468

DMSO). Plates were shaken for 3 min to homogenize the medium. Images of the plate were 469

acquired using the Scan^R screening station from Olympus. This platform includes an 470

epifluorescence microscope IX81 with a MT20 illumination system, an automated motorized 471

stage and a CCD camera. YFP fluorescence was recorded using the fluorescein isothiocyanate 472

parameters (YFP FITC, DAPI/FITC/TxRED filter cube, fluorescence illumination, FITC 473

492/18 excitation filter), the UPLSAPO 10x objective and an exposure time of 15 ms. Time-474

lapse movies were recorded with a resolution of 30 min between the pictures for up to 120 h. 475

Because cells need between 60-90 min to divide in optimal conditions, two to three images 476

were obtained capturing the mitotic figures of each cell. This time resolution allowed the 477

acquisition of four pictures for each of the 96 wells, covering about 10% of the well surfaces. 478

479

Screen of C17-Tolerant Mutants and Map-Based Cloning 480

To obtain mutants tolerant to C17, seeds from an ethylmethanesulphonate (EMS)-treated seed 481

collection with Col-0 background were plated on half-strength MS medium containing 2 μM 482

C17. After growing for seven days under long-day conditions (16 h of light/8 h of darkness) at 483

22°C, plants with a long root were identified as C17- tolerant mutants. Out of a total of 484

300,000 independent EMS-mutagenized seeds (divided over 20 pools), 22 mutants were 485

isolated (from 12 independent pools). To define the mutations underlying C17 tolerance, all 486

C17- tolerant mutants were backcrossed with wild-type (Col-0) plants to obtain F1 progenies. 487

F2 progenies from self-pollinated F1 plants were grown in the presence of 2 μM C17 for 488

17

seven days, allowing calculation of the segregation ratio of C17 tolerant within the F2 489

progenies. All mutants were used to generate mapping populations through crossing with 490

another ecotype (Ler-0). Simple sequence length polymorphism (SSLP) markers 491

(Supplemental Table 1) were used to map the position of the mutated genes in the Arabidopsis 492

genome. Subsequently, the mutant genes were identified through candidate gene sequencing. 493

For the cwm mutant screen, seeds of a set of confirmed T-DNA lines (CS27941) were 494

germinated on half-strength MS medium. Three-day-old seedlings were transferred to 495

medium with a low dose of C17 (200 nM), a concentration at which root growth is inhibited 496

instantly upon transfer to C17-containing medium. Two insertion lines (SALK_017325C and 497

SALK_020569C) were obtained with significant suppression of the growth inhibition in 498

presence of C17. Because the T-DNA insert within the AT5G44570 locus in SALK_020569C 499

was not linked with the C17- tolerant phenotype, SALK_020569C was crossed with the Ler-0 500

ecotype to generate a mapping population. SSLP markers (Supplemental Table 1) were used 501

to map the position of the mutated genes in the Arabidopsis genome. Subsequently, using 502

PCR, a second T-DNA insert was found and positioned at the coding region of AT1G32415. 503

504

Cellulose Measurement 505

The analysis of glucose content was performed on an AIR (Alcohol Insobuble Residue) 506

prepared as follows. One hundred mg (fresh weight) of ground 4-day-old dark-grown 507

seedlings were washed twice in four volumes of absolute ethanol for 15 min, then rinsed 508

twice in four volumes of acetone at room temperature for 10 min and left to dry under a fume 509

hood overnight at room temperature. AIR was then submitted to hydrolysis in 2.5 M 510

Trifluoroacetic acid (TFA) for 1.5 h at 100°C. To determine the cellulose content, the residual 511

pellet obtained after the TFA hydrolysis was rinsed twice with ten volumes of water and 512

hydrolysed with H2SO4 as described previously (Updegraff, 1969). The released glucose was 513

diluted 500 times and then quantified using a high performance anion exchange 514

chromatography with pulsed amperometric detection (HPAEC-PAD) as described previously 515

(Harholt et al., 2006). Quantification was done with biological quadruplicates and the average 516

values and standard errors are indicated in the graphs. 517

Detection of Cell Wall Weakening 518

For the detection of cell wall weakening, 3-day-old seedlings grown on half-strength MS 519

medium were transferred to liquid medium without or with 200 nM C17. The root tips were 520

stained with 10 mg mL-1 prodium iodide for three min. The stained root tips were put on the 521

18

Nunc™ Lab-Tek™ Chambered Coverglass (Catalog # 155361) without pressure or 522

microscope slide with cover slip that gave a gentle exerting of pressure. By using confocal 523

laser scanning microscopy (LSM710, ZEISS), the brittle cells could be visualized by the 524

uptake of PI. 525

Spinning Disk Microscopy and Image Analysis 526

For live cell imaging, hypocotyls of 3-day-old etiolated seedlings of the GFP-CESA3 line in 527

cesa3je5 treated without or with 50 µM C17 for 3 h were analysed on an Axiovert 200M 528

microscope (Zeiss) equipped with a Yokogawa CSU22 spinning disk, Zeiss 100/1.4 numerical 529

aperture oil objective, and Andor EMCCD iXon DU 895 camera (Plateforme d'Imagerie 530

Dynamique, Institut Pasteur, Paris, France). A 488- diode-pumped solid-state laser was used 531

for excitation, and emission was collected using band-pass 488/25 for GFP. For root tips 532

observation, 3-day-old light-grown seedlings were treated without or with the indicated drugs 533

and the change of GFP were observed with a time-series scanning from 0 min to 20 min. 534

535

Isolation of mitochondria from hydroponic cultures 536

Mitochondria were isolated from 14 day old hydroponically grown Arabidopsis plants as 537

previously described (Millar et al., 2001) with slight modifications. Plant material was 538

homogenized in grinding buffer (0.3 M sucrose, 25 mM tetrasodium pyrophosphate, 1% (w/v) 539

PVP-40, 2 mM EDTA, 10 mM KH2PO4, 1% (w/v) BSA, 20 mM ascorbic acid, pH 7.5) using 540

mortal and pestel for 2 to 5 min twice. The homogenate was filtered through four layers of 541

Miracloth and centrifuged at 2500 x g for 5 min, the resulting supernatant was then 542

centrifuged at 14,000 x g for 20 min. The resulting pellet was resuspended in sucrose wash 543

medium (0.3 M sucrose, 0.1% [w/v]) BSA, 10 mM TES (N-tris[hydroxymethyl]- methyl-2-544

aminoethanesulfonic acid], pH 7.5) and carefully layered over 35 ml PVP-40 gradient (30% 545

Percoll, 0 - 4% PVP). The gradient was centrifuged at 40 000 x g for 40 min. The 546

mitochondrial band was collected and washed 3 times in sucrose wash buffer without BSA by 547

20 000 x g for 20 min. 548

549

Oxygen consumption of whole seedlings and isolated mitochondria using an O2 electrode 550

Oxygen consumption of 6 days old Arabidopsis seedlings was measured using a computer-551

controlled Clark-type O2 electrode (Hansatech-Instruments, UK). All reactions were carried 552

19

out at 25 °C using 2 ml of whole tissue reaction medium (10 mM HEPES, 10 mM MES, 2 553

mM CaCl2, pH 7.2) and 40 - 60 mg plant material. To investigate AOX dependent respiration 554

rate, 1 mM of KCN was added. To inhibit AOX activity, 2 mM SHAM was added. 555

To measure oxygen uptake of isolated mitochondria, a 1 ml reaction medium (0.3 M 556

sucrose, 10 mM TES, 10 mM NaCl, 4 mM MgSO4, 0.1% (w/v) BSA, pH 7.2) together with 557

50 µg mitochondria protein was used. 5 mM succinate and 1 mM NADH were added. To 558

investigate the effect of C17 on isolated mitochondria, concentrations ranging from 1 to 8 µM 559

were added to mitochondria using succinate and NADH as substrates and compared to the 560

effect of 100 µM KCN as a cytochrome pathway inhibitor. 561

562

RNA Editing Analysis 563

For analysis of RNA editing, total RNA was isolated from the root tips (3-5 mm) of 5-day-old 564

seedlings using an RNeasy plant mini kit (Qiagen) and treated with DNase I (Invitrogen). 565

DNA-free RNA (2 μg) was reverse transcribed and sequences including the editing sites were 566

amplified by PCR. Primers to amplify the mitochondrial transcripts are described previously 567

(Bentolila et al., 2013). The RT-PCR products were sequenced immediately. 568

569

Mitochondria Isolation and Immunoblots 570

Two-week-old hydroponically grown Arabidopsis seedlings were used to isolate 571

mitochondria, according to the method described previously (Murcha and Whelan, 2015). 572

Mitochondrial proteins were separated by SDS-PAGE (Bio-Rad, Sydney) or blue-native gel 573

as described previously (Eubel et al., 2005), followed by transfer to Hybond-C extra 574

nitrocellulose (Bio-Rad, Sydney). Immunodetections were carried out as described previously 575

(Wang et al., 2012). To ensure linearity of detection, two dilutions of mitochondria were 576

loaded. Antibodies used were raised against Ndufs4 (Meyer et al., 2009), AOX (Elthon et al., 577

1989), Tim9 (Wang et al., 2012), Tim17 (Wang et al., 2012), RISP (Duncan et al., 2011) and 578

Tim23 (Wang et al., 2012). The antibodies against to Atpb (AS05 085), Cyt c (AS08 343A) 579

and COXII (AS04 0543A) were obtained from Agrisera. 580

For mitochondrial complexes staining, mitochondrial proteins (20 μg) were solubilized with 581

digitonin (5.0 g/g protein final) in digitonin extraction buffer (30 mM HEPES. 150 mM K-582

Acetate, 10% [v/v] glycerol, pH 7.4) and incubated on ice for 20 min. The samples were 583

centrifuged for 10 min at 15,000g, and Serva Blue G (0.2% [v/v] final) was added to the 584

20

supernatant. The samples were loaded onto NativePAGE Novex 4% to 16% Bis-Tris gels 585

(Life Technologies). Gels were washed twice for 10 min with distilled water and incubated in 586

complex I staining medium (0.1 M Tris pH7.4, 0.14 mM NADH, 1 mg ml-1 Nitro tetrazolium 587

blue), and in Peroxidase staining medium (10 mM phosphate buffer pH6.0, 20 mM guaiacol, 588

0.03% H2O2). After 2–3 h of staining, gels were transferred to Coomassie-colloidal fixing 589

solution (40% methanol, 10% acetic acid) to stop the reactions. 590

591

Accession Numbers 592

Sequence data from this article can be found in the Arabidopsis Genome Initiative or 593

GenBank/EMBL databases under the following accession numbers: CWM1 (AT1G17630), 594

CWM2 (AT1G32415), CESA1 (AT4G32410), CESA3 (AT5G05170), ANAC017 595

(AT1G34190), and SLO2 (AT2G13600). 596

597

Supplemental Data 598

The following materials are available in the online version of this article. 599

Supplemental video 1. Time-lapse imaging of Arabidopsis H2B-YFP suspension cells in 600 absence of C17. 601

Supplemental video 2. Time-lapse imaging of Arabidopsis H2B-YFP suspension cells in 602 the presence of 50 µM C17. 603

Supplemental Figure 1. Rough map position on the Arabidopsis genome of the mutated 604 genes rendering C17 tolerance. 605

Supplemental Figure 2. Sequence alignment of CESA1 (A) and CESA3 (B) of several 606 plant species. 607

Supplemental Figure 3. C17 does not trigger microtubule polymerization. 608

Supplemental Figure 4. C17 results in the depletion of CSCs from the root plasma 609 membrane. 610

Supplemental Figure 5. Isolation of cwm1 and cwm2 mutants. 611

Supplemental Figure 6. Both cwm1-1 and cwm2-1 mutations counteract the growth 612 inhibition induced by cellulose deficiency. 613

Supplemental Figure 7. The staining of respiratory protein complexes from wild type, 614 cwm1 and cwm2 mutants. 615

Supplemental Figure 8. Respiration rates of cwm1 and cwm2 mutants. 616

21

Supplemental Figure 9. C17 sensitivity of the mutants with defective mitochondrial 617 complex I. 618

Supplemental Figure 10. Inhibition of mitochondrial activity did not restore CSC activity. 619

Supplemental Figure 11. C17 does not directly inhibit isolated mitochondrial respiration. 620

ACKNOWLEDGMENTS 621

The authors thank Annick Bleys for help in preparing the manuscript. This work was 622

supported by the Integrated Project AGRONOMICS, in the Sixth Framework Programme of 623

the European Commission (LSHG-CT-2006-037704), and the Interuniversity Attraction Poles 624

Programme (IUAP P7/29 "MARS"), initiated by the Belgian Science Policy Office. T.C. and 625

I.D.C. are Postdoctoral Fellows of the Research Foundation-Flanders. I.D.C. is also supported 626

by FWO travel grant 12N2415N. F.V.B is supported by grants from the Interuniversity 627

Attraction Poles Programme (IUAP P7/29 ‘MARS’) initiated by the Belgian Science Policy 628

Office and Ghent University (Multidisciplinary Research Partnership ‘Biotechnology for a 629

Sustainable Economy’, grant 01MRB510W). A.H.M, K.B. Y.W, I.S and J.W were funded by 630

the ARC Centre of Excellence Plant Energy Biology (CE140100008). 631

632

AUTHOR CONTRIBUTIONS 633

Z.H., J.W., H.H., and L.D.V. conceived and designed the research. Z.H., R.V., T.C., Y.W. 634

I.D.C., O.L., K.B., G.M. and S.V. performed the experiments. Z.H., I.S., A.H.M., S.V. 635

F.V.B., J.W., H.H., and L.D.V. analysed the data. P.H. provided the platform for high-636

throughput chemical screen. Z.H. and L.D.V. wrote the article. All authors read, revised, and 637

approved the article. 638

639

REFERENCES 640

Barkan, A., and Small, I. (2014). Pentatricopeptide repeat proteins in plants. Annu. Rev. 641 Plant Biol. 65, 415-442. 642

Bartoli, C.G., Gómez, F., Martínez, D.E., and Guiamet, J.J. (2004). Mitochondria are the 643 main target for oxidative damage in leaves of wheat (Triticum aestivum L.). J. Exp. 644 Bot. 55, 1663-1669. 645

Beeckman, T., Przemeck, G.K.H., Stamatiou, G., Lau, R., Terryn, N., De Rycke, R., 646 Inzé, D., and Berleth, T. (2002). Genetic complexity of cellulose synthase A gene 647 function in Arabidopsis embryogenesis. Plant Physiol. 130, 1883-1893. 648

22

Bentolila, S., Knight, W., and Hanson, M. (2010). Natural variation in Arabidopsis leads to 649 the identification of REME1, a pentatricopeptide repeat-DYW protein controlling the 650 editing of mitochondrial transcripts. Plant Physiol. 154, 1966-1982. 651

Bentolila, S., Oh, J., Hanson, M.R., and Bukowski, R. (2013). Comprehensive high-652 resolution analysis of the role of an Arabidopsis gene family in RNA editing. PLoS 653 Genet. 9, e1003584. 654

Bleier, L., and Dröse, S. (2013). Superoxide generation by complex III: from mechanistic 655 rationales to functional consequences. Biochim. Biophys. Acta - Bioenerg. 1827, 656 1320-1331. 657

Boisnard-Lorig, C., Colon-Carmona, A., Bauch, W., Hodge, S., Doerner, P., Bancharel, 658 E., Dumas, C., Haseloff, J., and Berger, F. (2001). Dynamic analyses of the 659 expression of the HISTONE::YFP fusion protein in Arabidopsis show that syncytial 660 endosperm is divided in mitotic domains. Plant Cell 13, 495-509. 661

Brabham, C., Lei, L., Gu, Y., Stork, J., Barrett, M., and DeBolt, S. (2014). Indaziflam 662 herbicidal action: a potent cellulose biosynthesis inhibitor. Plant Physiol 166, 1177-663 1185. 664

Bray, E.A. (2004). Genes commonly regulated by water-deficit stress in Arabidopsis 665 thaliana. J. Exp. Bot. 55, 2331-2341. 666

Caño-Delgado, A., Penfield, S., Smith, C., Catley, M., and Bevan, M. (2003). Reduced 667 cellulose synthesis invokes lignification and defense responses in Arabidopsis 668 thaliana. Plant J. 34, 351-362. 669

Caño-Delgado, A.I., Metzlaff, K., and Bevan, M.W. (2000). The eli1 mutation reveals a 670 link between cell expansion and secondary cell wall formation in Arabidopsis 671 thaliana. Development 127, 3395-3405. 672

Clough, S.J., and Bent, A.F. (1998). Floral dip: a simplified method for Agrobacterium-673 mediated transformation of Arabidopsis thaliana. Plant J. 16, 735-743. 674

Crowell, E.F., Bischoff, V., Desprez, T., Rolland, A., Stierhof, Y.-D., Schumacher, K., 675 Gonneau, M., Höfte, H., and Vernhettes, S. (2009). Pausing of golgi bodies on 676 microtubules regulates secretion of cellulose synthase complexes in Arabidopsis. Plant 677 Cell 21, 1141-1154. 678

De Clercq, I., Vermeirssen, V., Van Aken, O., Vandepoele, K., Murcha, M.W., Law, 679 S.R., Inzé, A., Ng, S., Ivanova, A., Rombaut, D., van de Cotte, B., Jaspers, P., Van 680 de Peer, Y., Kangasjärvi, J., Whelan, J., and Van Breusegem, F. (2013). The 681 membrane-bound NAC transcription factor ANAC013 functions in mitochondrial 682 retrograde regulation of the oxidative stress response in Arabidopsis. Plant Cell 25, 683 3472-3490. 684

de Longevialle, A.F., Meyer, E.H., Andres, C., Taylor, N.L., Lurin, C., Millar, A.H., and 685 Small, I.D. (2007). The pentatricopeptide repeat gene OTP43 is required for trans-686 splicing of the mitochondrial nad1 Intron 1 in Arabidopsis thaliana. Plant Cell 19, 687 3256-3265. 688

Deng, Y., Srivastava, R., and Howell, S.H. (2013). Endoplasmic reticulum (ER) stress 689 response and its physiological roles in plants. Int. J. Mol. Sci. 14, 8188-8212. 690

Desprez, T., Vernhettes, S., Fagard, M., Refrégier, G., Desnos, T., Aletti, E., Py, N., 691 Pelletier, S., and Höfte, H. (2002). Resistance against herbicide isoxaben and 692 cellulose deficiency caused by distinct mutations in same cellulose synthase isoform 693 CESA6. Plant Physiol. 128, 482-490. 694

Desprez, T., Juraniec, M., Crowell, E.F., Jouy, H., Pochylova, Z., Parcy, F., Höfte, H., 695 Gonneau, M., and Vernhettes, S. (2007). Organization of cellulose synthase 696 complexes involved in primary cell wall synthesis in Arabidopsis thaliana. Proc. Natl. 697 Acad. Sci. USA 104, 15572-15577. 698

23

Duncan, O., Taylor, N.L., Carrie, C., Eubel, H., Kubiszewski-Jakubiak, S., Zhang, B., 699 Narsai, R., Millar, A.H., and Whelan, J. (2011). Multiple lines of evidence localize 700 signaling, morphology, and lipid biosynthesis machinery to the mitochondrial outer 701 membrane of Arabidopsis. Plant Physiol. 157, 1093-1113. 702

Ellis, C., and Turner, J.G. (2001). The Arabidopsis mutant cev1 has constitutively active 703 jasmonate and ethylene signal pathways and enhanced resistance to pathogens. Plant 704 Cell 13, 1025-1033. 705

Ellis, C., Karafyllidis, I., Wasternack, C., and Turner, J.G. (2002). The Arabidopsis 706 mutant cev1 links cell wall signaling to jasmonate and ethylene responses. Plant Cell 707 14, 1557-1566. 708

Elthon, T.E., Nickels, R.L., and McIntosh, L. (1989). Monoclonal antibodies to the 709 alternative oxidase of higher plant mitochondria. Plant Physiol. 89, 1311-1317. 710

Endler, A., and Persson, S. (2011). Cellulose synthases and synthesis in Arabidopsis. Mol. 711 Plant 4, 199-211. 712

Eubel, H., Braun, H.-P., and Millar, A.H. (2005). Blue-native PAGE in plants: a tool in 713 analysis of protein-protein interactions. Plant Methods 1, 11. 714

Francs-Small, C.C.d., Kroeger, T., Zmudjak, M., Ostersetzer-Biran, O., Rahimi, N., 715 Small, I., and Barkan, A. (2012). A PORR domain protein required for rpl2 and 716 ccmFC intron splicing and for the biogenesis of c-type cytochromes in Arabidopsis 717 mitochondria. Plant J. 69, 996-1005. 718

Fujita, M., Fujita, Y., Noutoshi, Y., Takahashi, F., Narusaka, Y., Yamaguchi-Shinozaki, 719 K., and Shinozaki, K. (2006). Crosstalk between abiotic and biotic stress responses: a 720 current view from the points of convergence in the stress signaling networks. Curr. 721 Opin. Plant Biol. 9, 436-442. 722

Gechev, T.S., Van Breusegem, F., Stone, J.M., Denev, I., and Laloi, C. (2006). Reactive 723 oxygen species as signals that modulate plant stress responses and programmed cell 724 death. BioEssays 28, 1091-1101. 725

Giraud, E., Van Aken, O., Ho, L.H.M., and Whelan, J. (2009). The transcription factor 726 ABI4 is a regulator of mitochondrial retrograde expression of ALTERNATIVE 727 OXIDASE1a. Plant Physiol. 150, 1286-1296. 728

Giraud, E., Ho, L.H.M., Clifton, R., Carroll, A., Estavillo, G., Tan, Y.-F., Howell, K.A., 729 Ivanova, A., Pogson, B.J., Millar, A.H., and Whelan, J. (2008). The absence of 730 ALTERNATIVE OXIDASE1a in Arabidopsis results in acute sensitivity to combined 731 light and drought stress. Plant Physiol. 147, 595-610. 732

Gutierrez, R., Lindeboom, J.J., Paredez, A.R., Emons, A.M.C., and Ehrhardt, D.W. 733 (2009). Arabidopsis cortical microtubules position cellulose synthase delivery to the 734 plasma membrane and interact with cellulose synthase trafficking compartments. Nat. 735 Cell Biol. 11, 797-806. 736

Hamann, T. (2015). The plant cell wall integrity maintenance mechanism—Concepts for 737 organization and mode of action. Plant Cell Physiol. 56, 215-223. 738

Harholt, J., Jensen, J.K., Sorensen, S.O., Orfila, C., Pauly, M., and Scheller, H.V. (2006). 739 ARABINAN DEFICIENT 1 is a putative arabinosyltransferase involved in 740 biosynthesis of pectic arabinan in Arabidopsis. Plant Physiol. 140, 49-58. 741

He, J., Duan, Y., Hua, D., Fan, G., Wang, L., Liu, Y., Chen, Z., Han, L., Qu, L.J., and 742 Gong, Z. (2012). DEXH box RNA helicase-mediated mitochondrial reactive oxygen 743 species production in Arabidopsis mediates crosstalk between abscisic acid and auxin 744 signaling. Plant Cell 24, 1815-1833. 745

Hématy, K., Sado, P.-E., Van Tuinen, A., Rochange, S., Desnos, T., Balzergue, S., 746 Pelletier, S., Renou, J.-P., and Höfte, H. (2007). A receptor-like kinase mediates the 747

24

response of Arabidopsis cells to the inhibition of cellulose synthesis. Curr. Biol. 17, 748 922-931. 749

Howell, S.H. (2013). Endoplasmic reticulum stress responses in plants. Annu. Rev. Plant 750 Biol. 64, 477-499. 751

Huang, S., Van Aken, O., Schwarzlander, M., Belt, K., and Millar, A.H. (2016). Roles of 752 mitochondrial reactive oxygen species in cellular signalling and stress response in 753 plants. Plant Physiol. 754

Iraki, N.M., Bressan, R.A., Hasegawa, P.M., and Carpita, N.C. (1989). Alteration of the 755 physical and chemical structure of the primary cell wall of growth-limited plant cells 756 adapted to osmotic stress. Plant Physiol. 91, 39-47. 757

Karimi, M., Inzé, D., and Depicker, A. (2002). GATEWAYTM vectors for Agrobacterium-758 mediated plant transformation. Trends Plant Sci. 7, 193-195. 759

Koprivova, A., des Francs-Small, C.C., Calder, G., Mugford, S.T., Tanz, S., Lee, B.R., 760 Zechmann, B., Small, I., and Kopriva, S. (2010). Identification of a 761 pentatricopeptide repeat protein implicated in splicing of intron 1 of mitochondrial 762 nad7 transcripts. The Journal of biological chemistry 285, 32192-32199. 763

Kranz, R.G., Richard-Fogal, C., Taylor, J.-S., and Frawley, E.R. (2009). Cytochrome c 764 biogenesis: mechanisms for covalent modifications and trafficking of heme and for 765 heme-iron redox control. Microbiol. Mol. Biol. Rev. 73, 510-528. 766

Lei, L., Singh, A., Bashline, L., Li, S., Yingling, Y.G., and Gu, Y. (2015). CELLULOSE 767 SYNTHASE INTERACTIVE1 is required for fast recycling of cellulose synthase 768 complexes to the plasma membrane in Arabidopsis. Plant Cell 27, 2926-2940. 769

Liu, Y., He, J., Chen, Z., Ren, X., Hong, X., and Gong, Z. (2010). ABA overly-sensitive 5 770 (ABO5), encoding a pentatricopeptide repeat protein required for cis-splicing of 771 mitochondrial nad2 intron 3, is involved in the abscisic acid response in Arabidopsis. 772 Plant J 63, 749-765. 773

Mavridou, D.A.I., Ferguson, S.J., and Stevens, J.M. (2013). Cytochrome c assembly. 774 IUBMB Life 65, 209-216. 775

May, M.J., and Leaver, C.J. (1993). Oxidative stimulation of glutathione synthesis in 776 Arabidopsis thaliana suspension cultures. Plant Physiol. 103, 621-627. 777

McFarlane, H.E., Döring, A., and Persson, S. (2014). The cell biology of cellulose 778 synthesis. Annu. Rev. Plant Biol. 65, 69-94. 779

Meyer, E.H., Tomaz, T., Carroll, A.J., Estavillo, G., Delannoy, E., Tanz, S.K., Small, 780 I.D., Pogson, B.J., and Millar, A.H. (2009). Remodeled respiration in ndufs4 with 781 low phosphorylation efficiency suppresses Arabidopsis germination and growth and 782 alters control of metabolism at night. Plant Physiol. 151, 603-619. 783

Miart, F., Desprez, T., Biot, E., Morin, H., Belcram, K., Höfte, H., Gonneau, M., and 784 Vernhettes, S. (2014). Spatio-temporal analysis of cellulose synthesis during cell 785 plate formation in Arabidopsis. Plant J. 77, 71-84. 786

Millar, A.H., Sweetlove, L.J., Giege, P., and Leaver, C.J. (2001). Analysis of the 787 Arabidopsis mitochondrial proteome. Plant Physiol 127, 1711-1727. 788

Millar, A.H., Whelan, J., Soole, K.L., and Day, D.A. (2011). Organization and regulation of 789 mitochondrial respiration in plants. Annu. Rev. Plant Biol. 62, 79-104. 790

Murashige, T., and Skoog, F. (1962). A revised medium for rapid growth and bio assays 791 with tobacco tissue cultures. Physiol. Plant. 15, 473-497. 792

Murcha, M.W., and Whelan, J. (2015). Isolation of intact mitochondria from the model 793 plant species Arabidopsis thaliana and Oryza sativa. Methods Mol. Biol. 1305, 1-12. 794

Ng, S., De Clercq, I., Van Aken, O., Law, S.R., Ivanova, A., Willems, P., Giraud, E., Van 795 Breusegem, F., and Whelan, J. (2014). Anterograde and retrograde regulation of 796

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nuclear genes encoding mitochondrial proteins during growth, development, and 797 stress. Mol. Plant 7, 1075-1093. 798

Ng, S., Ivanova, A., Duncan, O., Law, S.R., Van Aken, O., De Clercq, I., Wang, Y., 799 Carrie, C., Xu, L., Kmiec, B., Walker, H., Van Breusegem, F., Whelan, J., and 800 Giraud, E. (2013a). A membrane-bound NAC transcription factor, ANAC017, 801 mediates mitochondrial retrograde signaling in Arabidopsis. Plant Cell 25, 3450-3471. 802

Ng, S., Giraud, E., Duncan, O., Law, S.R., Wang, Y., Xu, L., Narsai, R., Carrie, C., 803 Walker, H., Day, D.A., Blanco, N.E., Strand, Å., Whelan, J., and Ivanova, A. 804 (2013b). Cyclin-dependent kinase E1 (CDKE1) provides a cellular switch in plants 805 between growth and stress responses. J. Biol. Chem. 288, 3449-3459. 806

Persson, S., Paredez, A., Carroll, A., Palsdottir, H., Doblin, M., Poindexter, P., Khitrov, 807 N., Auer, M., and Somerville, C.R. (2007). Genetic evidence for three unique 808 components in primary cell-wall cellulose synthase complexes in Arabidopsis. Proc. 809 Natl. Acad. Sci. USA 104, 15566-15571. 810

Piro, G., Leucci, M.R., Waldron, K., and Dalessandro, G. (2003). Exposure to water stress 811 causes changes in the biosynthesis of cell wall polysaccharides in roots of wheat 812 cultivars varying in drought tolerance. Plant Sci. 165, 559-569. 813

Rhoads, D.M., and Subbaiah, C.C. (2007). Mitochondrial retrograde regulation in plants. 814 Mitochondrion 7, 177-194. 815

Rhoads, D.M., Umbach, A.L., Subbaiah, C.C., and Siedow, J.N. (2006). Mitochondrial 816 reactive oxygen species. Contribution to oxidative stress and interorganellar signaling. 817 Plant Physiol. 141, 357-366. 818

Schmitz-Linneweber, C., and Small, I. (2008). Pentatricopeptide repeat proteins: a socket 819 set for organelle gene expression. Trends Plant Sci. 13, 663-670. 820

Skirycz, A., De Bodt, S., Obata, T., De Clercq, I., Claeys, H., De Rycke, R., Andriankaja, 821 M., Van Aken, O., Van Breusegem, F., Fernie, A.R., and Inze, D. (2010). 822 Developmental stage specificity and the role of mitochondrial metabolism in the 823 response of Arabidopsis leaves to prolonged mild osmotic stress. Plant Physiol 152, 824 226-244. 825

Srivastava, R., Deng, Y., Shah, S., Rao, A.G., and Howell, S.H. (2013). BINDING 826 PROTEIN is a master regulator of the endoplasmic reticulum stress sensor/transducer 827 bZIP28 in Arabidopsis. Plant Cell 25, 1416-1429. 828

Starkov, A.A. (2008). The role of mitochondria in reactive oxygen species metabolism and 829 signaling. Ann. NY Acad.Sci. 1147, 37-52. 830

Sung, T.-Y., Tseng, C.-C., and Hsieh, M.-H. (2010). The SLO1 PPR protein is required for 831 RNA editing at multiple sites with similar upstream sequences in Arabidopsis 832 mitochondria. Plant J. 63, 499-511. 833

Sweet, W.J., Morrison, J.C., Labavitch, J.M., and Matthews, M.A. (1990). Altered 834 synthesis and composition of cell wall of grape (Vitis vinifera L.) leaves during 835 expansion and growth-inhibiting water deficits. Plant Cell Physiol. 31, 407-414. 836

Takenaka, M. (2010). MEF9, an E-subclass pentatricopeptide repeat protein, is required for 837 an RNA editing event in the nad7 transcript in mitochondria of Arabidopsis. Plant 838 Physiol. 152, 939-947. 839

Takenaka, M., Verbitskiy, D., Zehrmann, A., and Brennicke, A. (2010). Reverse genetic 840 screening identifies five E-class PPR proteins involved in RNA editing in 841 mitochondria of Arabidopsis thaliana. J. Biol. Chem. 285, 27122-27129. 842

Tenhaken, R. (2015). Cell wall remodeling under abiotic stress. Front Plant Sci 5, 771. 843 Updegraff, D.M. (1969). Semimicro determination of cellulose in biological materials. Anal. 844

Biochem. 32, 420-424. 845

26

Van Aken, O., Zhang, B., Law, S., Narsai, R., and Whelan, J. (2013). AtWRKY40 and 846 AtWRKY63 modulate the expression of stress-responsive nuclear genes encoding 847 mitochondrial and chloroplast proteins. Plant Physiol. 162, 254-271. 848

Van Aken, O., De Clercq, I., Ivanova, A., Law, S.R., Van Breusegem, F., Millar, A.H., 849 and Whelan, J. (2016). Mitochondrial and chloroplast stress responses are modulated 850 in distinct touch and chemical inhibition phases in Arabidopsis. Plant Physiol. dx.doi.851 org/10.1104/pp.16.00273. 852

Vandavasi, V.G., Putnam, D.K., Zhang, Q., Petridis, L., Heller, W.T., Nixon, B.T., 853 Haigler, C.H., Kalluri, U., Coates, L., Langan, P., Smith, J.C., Meiler, J., and 854 O'Neill, H. (2016). A Structural Study of CESA1 Catalytic Domain of Arabidopsis 855 Cellulose Synthesis Complex: Evidence for CESA Trimers. Plant Physiol 170, 123-856 135. 857

Vanderauwera, S., Vandenbroucke, K., Inze, A., van de Cotte, B., Muhlenbock, P., De 858 Rycke, R., Naouar, N., Van Gaever, T., Van Montagu, M.C., and Van 859 Breusegem, F. (2012). AtWRKY15 perturbation abolishes the mitochondrial stress 860 response that steers osmotic stress tolerance in Arabidopsis. Proc Natl Acad Sci U S A 861 109, 20113-20118. 862

Vanlerberghe, G.C. (2013). Alternative oxidase: a mitochondrial respiratory pathway to 863 maintain metabolic and signaling homeostasis during abiotic and biotic stress in 864 plants. Int. J. Mol. Sci. 14, 6805-6847. 865

Verbitskiy, D., Zehrmann, A., van der Merwe, J.A., Brennicke, A., and Takenaka, M. 866 (2010). The PPR protein encoded by the LOVASTATIN INSENSITIVE 1 gene is 867 involved in RNA editing at three sites in mitochondria of Arabidopsis thaliana. Plant 868 J. 61, 446-455. 869

Wang, Y., Carrie, C., Giraud, E., Elhafez, D., Narsai, R., Duncan, O., Whelan, J., and 870 Murcha, M.W. (2012). Dual location of the mitochondrial preprotein transporters 871 B14.7 and Tim23-2 in complex I and the TIM17:23 complex in Arabidopsis links 872 mitochondrial activity and biogenesis. Plant Cell 24, 2675-2695. 873

Welchen, E., Hildebrandt, T.M., Lewejohann, D., Gonzalez, D.H., and Braun, H.-P. 874 (2012). Lack of cytochrome c in Arabidopsis decreases stability of Complex IV and 875 modifies redox metabolism without affecting Complexes I and III. Biochim. Biophys. 876 Acta - Bioenerg. 1817, 990-1001. 877

Wolf, S., Hématy, K., and Höfte, H. (2012). Growth control and cell wall signaling in 878 plants. Annu. Rev. Plant Biol. 63, 381-407. 879

Wu, J., Sun, Y., Zhao, Y., Zhang, J., Luo, L., Li, M., Wang, J., Yu, H., Liu, G., Yang, L., 880 Xiong, G., Zhou, J.-M., Zuo, J., Wang, Y., and Li, J. (2015). Deficient plastidic 881 fatty acid synthesis triggers cell death by modulating mitochondrial reactive oxygen 882 species. Cell Res. 25, 621-633. 883

Xia, Y., Lei, L., Brabham, C., Stork, J., Strickland, J., Ladak, A., Gu, Y., Wallace, I., 884 and DeBolt, S. (2014). Acetobixan, an inhibitor of cellulose synthesis identified by 885 microbial bioprospecting. PLoS ONE 9, e95245. 886

Yang, L., Zhang, J., He, J., Qin, Y., Hua, D., Duan, Y., Chen, Z., and Gong, Z. (2014). 887 ABA-mediated ROS in mitochondria regulate root meristem activity by controlling 888 PLETHORA expression in Arabidopsis. PLoS Genet 10, e1004791. 889

Zehrmann, A., Verbitskiy, D., van der Merwe, J.A., Brennicke, A., and Takenaka, M. 890 (2009). A DYW domain-containing pentatricopeptide repeat protein is required for 891 RNA editing at multiple sites in mitochondria of Arabidopsis thaliana. Plant Cell 21, 892 558-567. 893

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Zhu, J., Lee, B.H., Dellinger, M., Cui, X., Zhang, C., Wu, S., Nothnagel, E.A., and Zhu, 894 J.K. (2010). A cellulose synthase-like protein is required for osmotic stress tolerance 895 in Arabidopsis. Plant J 63, 128-140. 896

Zhu, Q., Dugardeyn, J., Zhang, C., Muhlenbock, P., Eastmond, P.J., Valcke, R., De 897 Coninck, B., Oden, S., Karampelias, M., Cammue, B.P., Prinsen, E., and Van Der 898 Straeten, D. (2014). The Arabidopsis thaliana RNA editing factor SLO2, which 899 affects the mitochondrial electron transport chain, participates in multiple stress and 900 hormone responses. Mol Plant 7, 290-310. 901

Zsigmond, L., Rigo, G., Szarka, A., Szekely, G., Otvos, K., Darula, Z., Medzihradszky, 902 K.F., Koncz, C., Koncz, Z., and Szabados, L. (2008). Arabidopsis PPR40 connects 903 abiotic stress responses to mitochondrial electron transport. Plant Physiol 146, 1721-904 1737. 905

906

907

908 909 910

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LEGENDS TO FIGURES 911

Figure 1. C17 interferes with cytokinesis. (A) C17 chemical structure (ChemDiv, Catalogue#: 912

7693622). (B) H2B-YFP-labelled nuclei of Arabidopsis suspension cells in absence (mock) or 913

presence (C17) of 50 µM C17 for 72 h. (C) The five main phases (interphase, prophase, 914

metaphase, anaphase, and telophase) of mitosis of suspension cells cultivated under control 915

conditions (mock, upper panel) or in the presence of 50 µM C17 (C17, bottom panel). 916

Figure 2. C17-tolerant mutants. (A) Five-day-old wild-type (Col-0) seedlings grown with (0.1 917

µM, 0.2 µM, 0.5 µM, 1 µM, 2 µM or 5 µM) or without (mock) C17. (B) Quantification of the 918

root length of seedlings shown in (A). Data represent mean ± SD (n > 10). Statistically 919

significant differences compared with wild-type plants in absence of C17 are indicated, *P-920

value < 0.01 (two-sided Student’s t-test). (C-D) Roots of 5-day-old wild type (Col-0) and 22 921

C17-tolerant mutants grown in the absence (C) or presence (D) of 2 µM C17. Scale bars = 5 922

mm. 923

Figure 3. Mapping of mutations rendering C17 tolerance. (A-B) Genetic mapping and gene 924

structure of CESA1 (A) and CESA3 (B). The cesa17l and cesa32c loci were mapped to CESA1 925

(AT4G32410) and CESA3 (AT5G05170), respectively. The gene structure is shown at the 926

lower panel: exons are represented as filled rectangles, introns are shown as lines. The 927

nucleotide replacement in the mutant allele is indicated. Scale bar = 100 kb. (C) Schematic 928

diagram of the domains and mutation locations in CESA1 and CESA3. CESA1 and CESA3 929

proteins are located in the plasma membrane with eight predicted transmembrane domains 930

(black boxes). The C17-tolerant mutations are indicated by coloured stars. The corresponding 931

amino acid changes are listed. 932

Figure 4. C17 inhibits cellulose biosynthesis and depletes CSCs from the plasma membrane. 933

(A) Hypocotyl elongation of 5-day-old dark-grown wild type (Col-0, left panel) and a C17-934

tolerant mutant (cesa1A1018V, right panel) in the absence (mock) or presence of C17 (0.05 µM, 935

0.1 µM, 0.2 µM and 0.5 µM). Scale bars = 0.25 mm. (B) Quantification of the hypocotyl 936

length of plants shown in (A). Data represent mean ± SD (n > 10). Statistically significant 937

differences compared with wild-type plants are indicated, *P-value < 0.01 (two-sided 938

Student’s t-test). (C) Glucose content of the hypocotyl of 5-day-old dark-grown wild type in 939

the presence of C17 (0 µM, 0.1 µM and 0.2 µM). Data represent mean ± SD (n = 4). 940

Statistically significant differences compared with wild-type plants are indicated, *P-value < 941

0.01 (two-sided Student’s t-test). (D) GFP-CESA3 localization at the plasma membrane in the 942

29

absence (mock) or presence of 50 µM C17 for 2 h. Single optical sections and time averages 943

of 61 frames (5-min duration in 5-s intervals) of plasma membrane-localized GFP-CESA3. 944

Scale bars = 10 µm. 945

Figure 5. C17 results in a brittle cell wall. (A) Representative confocal microscopy images of 946

plants stained with propidium iodide (PI). Four-day-old wild-type (Col-0, left panel) and C17 947

tolerant mutant (cesa1A1018V, right panel) seedlings were treated with 200 nM C17 for 0 h, 1 h, 948

2 h or 3 h and roots were collected and stained with PI. The broken cells with brittle cell wall 949

were visualized by the uptake of PI. Scale bars = 50 µm. (B) Representative confocal 950

microscopy images of 4-day-old wild-type and je5 mutant seedlings with a weak allele of 951

CESA3 stained with PI. Scale bar = 50 µm. (C) Root growth of 4-day-old wild-type (Col-0) 952

and C17-tolerant mutant (cesa1A108V) seedlings in the presence of 200 nM C17. Data represent 953

mean ± SD (n > 5). Statistically significant differences compared with wild-type plants are 954

indicated, *P-value < 0.01 (two-sided Student’s t-test). 955

Figure 6. Suppression of C17 sensitivity by the deficiency of CWM1 and CWM2. (A) Root 956

growth of wild type (Col-0), cwm1 and cwm2 mutants. Three-day-old seedlings grown on 957

half-strength MS medium were transferred for two days to control medium (mock) or medium 958

containing 200 nM C17. Arrowheads indicate the root tip position at the moment of transfer. 959

Scale bars = 5 mm. (B) Quantification of root elongation of plants after transfer. Data 960

represent mean ± SD (n > 10). Statistically significant differences compared with wild-type 961

plants are indicated, *P-value < 0.01 (two-sided Student’s t-test). 962

Figure 7. Both CWM1 and CWM2 are involved in mitochondrial RNA editing events. (A) 963

Multiple mitochondrial RNA editing defects in cwm1 mutants. Sequencing chromatograms of 964

editing sites (ccmB-428, nad5-598 and ccmC-463) of wild type (left panel), cwm1-1 (middle 965

panel) and cwm1-2 (right panel) are displayed. (B) ccmC-575 editing defect in cwm2 mutants. 966

Sequencing chromatograms of the ccmC-575 editing site of wild type (left panel), cwm2-1 967

(middle panel) and cwm2-2 (right panel) are displayed. The editing sites are marked with light 968

blue colour; amino acid changes by editing defects are listed on the left. 969

Figure 8. Deficiency of CWM1 and CWM2 perturbs mitochondrial function. (A) 970

Mitochondrial protein content was quantified using antibodies. Antibodies used detect ATP 971

synthase (Atpb, subunit of complex V), ubiquinone oxidoreductase Fe-S protein4 (NDUFS4, 972

subunit of complex I), Rieske iron– 29 ulphur protein (RISP, subunit of complex III), 973

cytochrome oxidase subunit II (COXII, subunit of complex IV), cytochrome c (Cyt c), 974

30

alternative oxidase (AOX) and mitochondrial import inner membrane translocase subunits 975

(Tim 9, Tim 17 and Tim 23). (B) Image of total mitochondrial proteins of wild type (WT), 976

cwm1 mutants (cwm1-1 and cwm1-2) and cwm2 mutants (cwm2-1 and cwm2-2). 2 µg or 4 µg 977

of mitochondrial proteins were separated with SDS-PAGE and stained with coomassie blue. 978

(C) Mitochondrial complexes in wild type (WT), cwm1 mutants (cwm1-1 and cwm1-2) and 979

cwm2 mutants (cwm2-1 and cwm2-2). Mitochondrial proteins were separated with blue native 980

polyacrylamide gel electrophoresis (BN-PAGE). Atpb antibody is used for complex V (C), 981

RISP for complex III (D), COXII for complex IV (E), and NDUFS4 for complex I (F). The 982

identities of protein complexes are indicated on the left or right side of the blots: I – complex 983

I; IV – complex IV; V – complex V; III2 – dimeric complex III; I+III2 supercomplex 984

composed of complex I and dimeric complex III; I2+III4 – a dimer of supercomplex I+III2. 985

Figure 9. Inhibition of mitochondrial complex III phenocopies the C17-tolerance phenotype 986

of cwm1 and cwm2 mutants. (A) Root elongation of wild-type control-treated (DMSO), 1 µM 987

antimycin A-treated (AA), and 50 µM rotenone-treated (RO) plants. Three-day-old seedlings 988

grown on half-strength MS medium were transferred to medium without (left panel) or with 989

(right panel) 200 nM C17 for 2 days. Arrowheads indicate the root tip position at the moment 990

of transfer. Scale bars = 5 mm. (B) Quantification of the root elongation of plants after 991

transfer. Data represent mean ± SD (n > 10). Statistically significant differences compared 992

with wild-type plants in the absence of mitochondrial inhibitors are indicated, *P-value < 0.01 993

(two-sided Student’s t-test). (C) Representative confocal microscopy images of 4-day-old 994

wild type (Col-0) control-treated with 0.1% DMSO (mock), or with 1 µM antimycin A (AA), 995

200 nM C17 (C17), or a combination of 1 µM antimycin A with 200 nM C17 (C17+AA). 996

Two-hour treated roots were stained with PI. The brittle cell wall was visualized by the uptake 997

of PI. Scale bar = 100 µm. (D-F) Representative spinning confocal microscopy images of 4-998

day-old GFP-CESA3 plants treated with 0.1% DMSO (D), 200 nM C17 (E), or a combination 999

of 1 µM antimycin A with 200 nM C17 (F). The images were taken at 20 min after treatment. 1000

Scale bars = 5 µm. (G) Quantification of fluorescence in D-F. The relative intensity is 1001

calculated by the fluorescence per unit area in root elongation zone of each sample dividing 1002

that of wild-type plants in mock. Data represent mean ± SD (n = 5). Statistically significant 1003

differences compared with wild-type plants in mock are indicated, *P-value < 0.01 (two-sided 1004

Student’s t-test). 1005

Figure 10. Both cwm1 and cwm2 Mutations Enhance the Tolerance of je5 against Osmotic 1006

Stress. (A) Relative 2-day elongation of wild type (Col-0) after transferring to the medium 1007

31

without (Mock) and with 200 mM mannitol (Mannitol), 100 nM C17 (C17) and the 1008

combination of 200 mM mannitol and 100 nM C17 (Mannitol+C17). Data represent mean ± 1009

SD (n > 10). Statistically significant differences compared with Mock are indicated, *P < 0.01 1010

(two-sided Student’s t-test). (B) Root growth of je5, je5 cwm1 and je5 cwm2 mutants in 1011

presence of 250 mM mannitol. Three-day-old seedlings grown on half-strength MS medium 1012

were transferred to medium with 250 mM mannitol for 2 days; Arrowheads indicate root tip 1013

position at the moment of transfer. Scale bar = 2.5 mm. (C Relative 2-day elongation of wild 1014

type (Col-0), cwm1-1, cwm2-1, je5, je5 cwm1-1 and je5 cwm2-1 after transferring to the 1015

medium supplemented with 250 mM mannitol. Data represent mean ± SD (n > 10). 1016

Statistically significant differences compared with wild-type plants are indicated, *P < 0.01 1017

(two-sided Student’s t-test). 1018

Figure 11. ANAC017 is a component linking mitochondria and cell wall. (A) Root growth of 1019

wild type (Col-0) and ANAC017 overexpressing lines (ANAC017OE-2 and ANAC017OE-16) in 1020

the presence of 200 nM C17. Three-day-old seedlings grown on half-strength MS medium 1021

were transferred to medium with 200 nM C17 for two days. Arrowheads indicate the root tip 1022

position at the moment of transfer. Scale bar = 2.5 mm. (B) Relative root elongation of wild 1023

type (Col-0) and ANAC017 overexpressing lines (ANAC017OE-2 and ANAC017OE-16). Three-1024

day-old seedlings grown on half-strength MS medium were transferred to medium without 1025

(mock) or with 200 nM C17 for two days. Data represent mean ± SD (n > 10). (C) Root 1026

growth of wild type (Col-0), ANAC017 knockout mutant (anac017-1), cwm2-1 single mutant 1027

and cwm2-1 anac017-1 double mutant in the presence of 200 nM C17. Three-day-old 1028

seedlings grown on half-strength MS medium were transferred to medium with 200 nM C17 1029

for two days. Arrowheads indicate the root tip position at the moment of transfer. (D-G) 1030

Representative images of root mature zone of wild type (Col-0, D), ANAC017 knockout 1031

mutant (anac017-1, E), cwm2-1 single mutant (F) and cwm2-1 anac017-1 double mutant (G) 1032

in the presence of 200 nM C17. Scale bar = 50µm. (H) Relative root elongation of wild type 1033

(Col-0), ANAC017 knockout mutant (anac017-1), cwm2-1 single mutant and cwm2-1 1034

anac017-1 double mutant in the presence of 200 nM C17. Three-day-old seedlings grown on 1035

half-strength MS medium were transferred to medium without (mock) or with 200 nM C17 1036

for two days. Data represent mean ± SD (n > 10). (I) The length of mature cortical cells of 1037

wild type (Col-0), ANAC017 knockout mutant (anac017-1), cwm2-1 single mutant and cwm2-1038

1 anac017-1 double mutant in the presence of 200 nM C17. Data represent mean ± SD (n > 1039

32

20). Statistically significant differences are indicated, *P-value < 0.01 (two-sided Student’s t-1040

test). 1041

Supplemental Data 1042

Supplemental Figure 1. Rough map position on the Arabidopsis genome of the mutated 1043

genes rendering C17 tolerance. By using 24 general simple sequence length polymorphism 1044

(SSLP) markers, the mutated genes in C17-tolerant mutants were mapped to Arabidopsis 1045

genome. The mutated genes are divided into two groups based on the linkage with SSLP 1046

markers, which were designated by corresponding markers, CH4-14494 and CH5-512, 1047

respectively. 1048

Supplemental Figure 2. Sequence alignment of CESA1 (A) and CESA3 (B) of several plant 1049

species. Sequences were aligned with a multiple sequence alignment programme 1050

(http://www.genome.jp/tools/clustalw/) using CLUSTALW algorithms. Protein database 1051

accession numbers are: CESA1A.thaliana-NP_194967; CESA1G.max-XP_003522623; 1052

CESA1F.vesca-XP_004291468; CESA1V.vinifera-XP_002282575; CESA1S.lycopersicum-1053

XP_004245031; CESA1Z.mays-NP_001104954; CESA1O.sativa-NP_001054788; CESA3A.thaliana-1054

NP_196136; CESA3G.max-XP_003540527; CESA3F.vesca-XP_004306536; CESA3V.vinifera-1055

XP_002278997; CESA3S.lycopersicum-XP_004229630; CESA3Z.mays-NP_001105621; 1056

CESA3O.sativa-NP_001059162. The amino acid regions harbouring amino acid replacements in 1057

C17-tolerant mutants were selected. Arrowheads indicate the positions of the mutated amino 1058

acids. The blue lines indicate the predicted transmembrane domains (TM). 1059

Supplemental Figure 3. C17 does not trigger microtubule polymerization. GFP-CESA3 1060

localization at the plasma membrane and cortical microtubule arrays were observed in roots in 1061

absence (mock) or presence of C17 (200 nM) for 30 min. Transgenic plants harboring both 1062

GFP-CESA3 and MBD-Cherry were used. Scale bars = 5 µm. 1063

Supplemental Figure 4. C17 results in the depletion of CSCs from the root plasma 1064

membrane. Representative spinning confocal microscopy images of 4-day-old GFP-CESA3 1065

roots treated with C17 (200 nM). The images were taken at indicated time points (0 min, 5 1066

min, 10 min, 15 min and 20 min). Depletion of GFP-CESA3 is indicated by loss of GFP 1067

fluorescence from the plasma membrane (indicated by the arrows). 1068

Supplemental Figure 5. Isolation of cwm1 and cwm2 mutants. (A) Intron-exon organization 1069

of the CWM1 gene. The black box represents the exon. The position of T-DNA insertion sites 1070

33

(cwm1-1, cwm1-2, and cwm1-3) are indicated. Scale bar = 200 bp. (B) The C17 sensitivity of 1071

two independent AT5G44570 knockout mutants lines (SALK_020569C and SAIL_699_C11). 1072

Three-day-old seedlings grown on half-strength MS medium were transferred to medium 1073

without (-) or with (+) 200 nM C17 for two days. Arrowheads indicate the root tip position at 1074

the moment of transfer. Scale bar = 5 mm. (C) Mapping and gene structure of CWM2. The 1075

cwm2-1 locus was mapped into the 171.8-kb region between SSLP markers CH1_11687 and 1076

CH1_11589. Using PCR, a second T-DNA insert was found and positioned at the coding 1077

region of AT1G32415. The black box represents the exon. The position of the T-DNA 1078

insertion sites (cwm2-1 and cwm2-2) are indicated. 1079

Supplemental Figure 6. Both cwm1-1 and cwm2-1 mutations counteract the growth 1080

inhibition induced by cellulose deficiency. Three-day-old seedlings grown on half-strength 1081

MS medium were transferred to medium without (mock, left panel), with isoxaben (4 nM 1082

panel) or with indaziflam (0.2 nM, right panel). Arrowheads indicate the root tip position at 1083

the moment of transfer. Scale bar = 5 mm. 1084

Supplemental Figure 7. The staining of respiratory protein complexes from wild type, cwm1 1085

and cwm2 mutants. Mitochondria proteins were isolated from two-week old seedlings and 1086

solubilized by digitonin extraction buffer, separated by BN-PAGE and either visualized by 1087

Coomassie staining or by in-gel activity staining for complex I and complex IV. The identities 1088

of protein complexes are indicated on the left or right side of the blots: I – complex I; IV – 1089

complex IV; V – complex V; III2 – dimeric complex III; I+III2 supercomplex composed of 1090

complex I and dimeric complex III; I2+III4 – a dimer of supercomplex I+III2. 1091

Supplemental Figure 8. Whole seedling respiration rates of cwm1 and cwm2 mutants. (A) 1092

Oxygen consumption of whole seedlings of Col-0, cwm1 and cwm2 mutants. Data represent 1093

mean ± SE (n = 4). (B) Relative KCN resistant respiration of whole seedlings of Col-0, cwm1 1094

and cwm2 mutants. Data represent mean ± SE (n = 4). Statistically significant differences 1095

compared with wild-type plants are indicated with P-value (two-sided Student’s t-test). 1096

Supplemental Figure 9. C17 sensitivity of the mutants with defective mitochondrial complex 1097

I. Root growth of wild-type (Col-0) and mitochondrial complex I mutants (ndsf4, bir6-2 and 1098

otp439). Three-day-old seedlings grown on half-strength MS medium were transferred to 1099

medium without (-) or with (+) of 200 nM C17 for two days. Arrowheads indicate the root tip 1100

position at the moment of transfer. Scale bar = 5 mm. 1101

34

Supplemental Figure 10. Inhibition of mitochondrial activity did not restore CSC activity. 1102

(A-F) Representative spinning confocal microscopy images of 4-day-old GFP-CESA3 plants 1103

treated with 0.1% DMSO (A and D), 200 nM C17 (B and E), or a combination of 1 µM 1104

antimycin A with 200 nM C17 (C and F). The images were taken at 0 min (A, B and C) and 1105

20 min (D, E and F). Depletion of GFP-CESA3 is indicated by loss of GFP fluorescence from 1106

the plasma membrane, such as the domains indicated by the arrows. Scale bars = 25 µm. 1107

Supplemental Figure 11. C17 does not directly inhibit isolated mitochondrial respiration. 1108

The oxygen uptake of isolated mitochondria was measure to evaluate the effect of C17 on 1109

mitochondria. Data represent mean ± SE (n = 4). C17 concentrations ranging from 1 to 8 µM 1110

were added to mitochondria using succinate (succ) and NADH as substrates. A cytochrome 1111

pathway inhibitor, KCN (100 µM) was used as positive control. 1112

1113

1114

1115

1116

1117

1118

1119

1120

35

Table 1: CESA1 and CESA3 alleles 1121

1122

C17-tolerant mutants Genetic property Gene mutated Mutation position 1B Semi-dominant CESA1 A1023T

3D Semi-dominant CESA1 V297M

3E, 4H, 7L, 17Y, 19B1 Semi-dominant CESA1 A1018V

3G, 9Q Recessive CESA1 L872F

3F Semi-dominant CESA1 S892N

9R Recessive CESA1 G1013R

14V Semi-dominant CESA1 K945R

14U, 17Z, 20D1 Recessive CESA1 G1013E

18A1 Recessive CESA1 S307L

20C1 Semi-dominant CESA1 P1010L

2C, 5R, 6K, 15W Semi-dominant CESA3 S983F

8P Semi-dominant CESA3 S1037F

Figure 1. C17 interferes with cytokinesis. (A) C17 chemical structure (ChemDiv,

Catalogue#: 7693622). (B) H2B-YFP-labelled nuclei of Arabidopsis suspension cells

in absence (mock) or presence (C17) of 50 µM C17 for 72 h. (C) The five main phases

(interphase, prophase, metaphase, anaphase, and telophase) of mitosis of suspension

cells cultivated under control conditions (mock, upper panel) or in the presence of 50

µM C17 (C17, bottom panel).

Figure 2. C17-tolerant mutants. (A) Five-day-old wild-type (Col-0) seedlings grown

with (0.1 µM, 0.2 µM, 0.5 µM, 1 µM, 2 µM or 5 µM) or without (mock) C17. (B)

Quantification of the root length of seedlings shown in (A). Data represent mean ± SD

(n > 10). Statistically significant differences compared with wild-type plants in absence

of C17 are indicated, *P-value < 0.01 (two-sided Student’s t-test). (C-D) Roots of 5-

day-old wild type (Col-0) and 22 C17-tolerant mutants grown in the absence (C) or

presence (D) of 2 µM C17. Scale bars = 5 mm.

Figure 3. Mapping of mutations rendering C17 tolerance. (A-B) Genetic mapping and

gene structure of CESA1 (A) and CESA3 (B). The cesa17l and cesa32c loci were mapped

to CESA1 (AT4G32410) and CESA3 (AT5G05170), respectively. The gene structure is

shown at the lower panel: exons are represented as filled rectangles, introns are shown

as lines. The nucleotide replacement in the mutant allele is indicated. Scale bar = 100

kb. (C) Schematic diagram of the domains and mutation locations in CESA1 and

CESA3. CESA1 and CESA3 proteins are located in the plasma membrane with eight

predicted transmembrane domains (black boxes). The C17-tolerant mutations are

indicated by coloured stars. The corresponding amino acid changes are listed.

Figure 4. C17 inhibits cellulose biosynthesis and depletes CSCs from the plasma

membrane. (A) Hypocotyl elongation of 5-day-old dark-grown wild type (Col-0, left

panel) and a C17-tolerant mutant (cesa1A1018V, right panel) in the absence (mock) or

presence of C17 (0.05 µM, 0.1 µM, 0.2 µM and 0.5 µM). Scale bars = 0.25 mm. (B)

Quantification of the hypocotyl length of plants shown in (A). Data represent mean ±

SD (n > 10). Statistically significant differences compared with wild-type plants are

indicated, *P-value < 0.01 (two-sided Student’s t-test). (C) Glucose content of the

hypocotyl of 5-day-old dark-grown wild type in the presence of C17 (0 µM, 0.1 µM

and 0.2 µM). Data represent mean ± SD (n = 4). Statistically significant differences

compared with wild-type plants are indicated, *P-value < 0.01 (two-sided Student’s t-

test). (D) GFP-CESA3 localization at the plasma membrane in the absence (mock) or

presence of 50 µM C17 for 2 h. Single optical sections and time averages of 61 frames

(5-min duration in 5-s intervals) of plasma membrane-localized GFP-CESA3. Scale

bars = 10 µm.

Figure 5. C17 results in a brittle cell wall. (A) Representative confocal microscopy

images of plants stained with propidium iodide (PI). Four-day-old wild-type (Col-0, left

panel) and C17 tolerant mutant (cesa1A1018V, right panel) seedlings were treated with

200 nM C17 for 0 h, 1 h, 2 h or 3 h and roots were collected and stained with PI. The

broken cells with brittle cell wall were visualized by the uptake of PI. Scale bars = 50

µm. (B) Representative confocal microscopy images of 4-day-old wild-type and je5

mutant seedlings with a weak allele of CESA3 stained with PI. Scale bar = 50 µm. (C)

Root growth of 4-day-old wild-type (Col-0) and C17-tolerant mutant (cesa1A108V)

seedlings in the presence of 200 nM C17. Data represent mean ± SD (n > 5). Statistically

significant differences compared with wild-type plants are indicated, *P-value < 0.01

(two-sided Student’s t-test).

Figure 6. Suppression of C17 sensitivity by the deficiency of CWM1 and CWM2. (A)

Root growth of wild type (Col-0), cwm1 and cwm2 mutants. Three-day-old seedlings

grown on half-strength MS medium were transferred for two days to control medium

(mock) or medium containing 200 nM C17. Arrowheads indicate the root tip position

at the moment of transfer. Scale bars = 5 mm. (B) Quantification of root elongation of

plants after transfer. Data represent mean ± SD (n > 10). Statistically significant

differences compared with wild-type plants are indicated, *P-value < 0.01 (two-sided

Student’s t-test).

Figure 7. Both CWM1 and CWM2 are involved in mitochondrial RNA editing events.

(A) Multiple mitochondrial RNA editing defects in cwm1 mutants. Sequencing

chromatograms of editing sites (ccmB-428, nad5-598 and ccmC-463) of wild type (left

panel), cwm1-1 (middle panel) and cwm1-2 (right panel) are displayed. (B) ccmC-575

editing defect in cwm2 mutants. Sequencing chromatograms of the ccmC-575 editing

site of wild type (left panel), cwm2-1 (middle panel) and cwm2-2 (right panel) are

displayed. The editing sites are marked with light blue colour; amino acid changes by

editing defects are listed on the left.

Figure 8. Deficiency of CWM1 and CWM2 perturbs mitochondrial function. (A)

Mitochondrial protein content was quantified using antibodies. Antibodies used detect

ATP synthase (Atpb, subunit of complex V), ubiquinone oxidoreductase Fe-S protein4

(NDUFS4, subunit of complex I), Rieske iron–1ulphur protein (RISP, subunit of

complex III), cytochrome oxidase subunit II (COXII, subunit of complex IV),

cytochrome c (Cyt c), alternative oxidase (AOX) and mitochondrial import inner

membrane translocase subunits (Tim 9, Tim 17 and Tim 23). (B) Image of total

mitochondrial proteins of wild type (WT), cwm1 mutants (cwm1-1 and cwm1-2) and

cwm2 mutants (cwm2-1 and cwm2-2). 2 µg or 4 µg of mitochondrial proteins were

separated with SDS-PAGE and stained with coomassie blue. (C) Mitochondrial

complexes in wild type (WT), cwm1 mutants (cwm1-1 and cwm1-2) and cwm2 mutants

(cwm2-1 and cwm2-2). Mitochondrial proteins were separated with blue native

polyacrylamide gel electrophoresis (BN-PAGE). Atpb antibody is used for complex V

(C), RISP for complex III (D), COXII for complex IV (E), and NDUFS4 for complex I

(F). The identities of protein complexes are indicated on the left or right side of the

blots: I – complex I; IV – complex IV; V – complex V; III 2 – dimeric complex III; I+III2

supercomplex composed of complex I and dimeric complex III; I2+III 4 – a dimer of

supercomplex I+III2.

Figure 9. Inhibition of mitochondrial complex III phenocopies the C17-tolerance

phenotype of cwm1 and cwm2 mutants. (A) Root elongation of wild-type control-

treated (DMSO), 1 µM antimycin A-treated (AA), and 50 µM rotenone-treated (RO)

plants. Three-day-old seedlings grown on half-strength MS medium were transferred

to medium without (left panel) or with (right panel) 200 nM C17 for 2 days.

Arrowheads indicate the root tip position at the moment of transfer. Scale bars = 5 mm.

(B) Quantification of the root elongation of plants after transfer. Data represent mean ±

SD (n > 10). Statistically significant differences compared with wild-type plants in the

absence of mitochondrial inhibitors are indicated, *P-value < 0.01 (two-sided Student’s

t-test). (C) Representative confocal microscopy images of 4-day-old wild type (Col-0)

control-treated with 0.1% DMSO (mock), or with 1 µM antimycin A (AA), 200 nM

C17 (C17), or a combination of 1 µM antimycin A with 200 nM C17 (C17+AA). Two-

hour treated roots were stained with PI. The brittle cell wall was visualized by the

uptake of PI. Scale bar = 100 µm. (D-F) Representative spinning confocal microscopy

images of 4-day-old GFP-CESA3 plants treated with 0.1% DMSO (D), 200 nM C17

(E), or a combination of 1 µM antimycin A with 200 nM C17 (F). The images were

taken at 20 min after treatment. Scale bars = 5 µm. (G) Quantification of fluorescence

in D-F. The relative intensity is calculated by the fluorescence per unit area in root

elongation zone of each sample dividing that of wild-type plants in mock. Data

represent mean ± SD (n = 5). Statistically significant differences compared with wild-

type plants in mock are indicated, *P-value < 0.01 (two-sided Student’s t-test).

Figure 10. Both cwm1 and cwm2 mutations enhance the tolerance of je5 against

osmotic stress. (A) Relative 2-day elongation of wild type (Col-0) after transferring to

the medium without (Mock) and with 200 mM mannitol (Mannitol), 100 nM C17 (C17)

and the combination of 200 mM mannitol and 100 nM C17 (Mannitol+C17). Data

represent mean ± SD (n > 10). Statistically significant differences compared with Mock

are indicated, *P < 0.01 (two-sided Student’s t-test). (B) Root growth of je5, je5 cwm1

and je5 cwm2 mutants in presence of 250 mM mannitol. Three-day-old seedlings grown

on half-strength MS medium were transferred to medium with 250 mM mannitol for 2

days; Arrowheads indicate root tip position at the moment of transfer. Scale bar = 2.5

mm. (C Relative 2-day elongation of wild type (Col-0), cwm1-1, cwm2-1, je5, je5

cwm1-1 and je5 cwm2-1 after transferring to the medium supplemented with 250 mM

mannitol. Data represent mean ± SD (n > 10). Statistically significant differences

compared with wild-type plants are indicated, *P < 0.01 (two-sided Student’s t-test).

Figure 11. ANAC017 is a component linking mitochondria and cell wall. (A) Root

growth of wild type (Col-0) and ANAC017 overexpressing lines (ANAC017OE-2 and

ANAC017OE-16) in the presence of 200 nM C17. Three-day-old seedlings grown on

half-strength MS medium were transferred to medium with 200 nM C17 for two days.

Arrowheads indicate the root tip position at the moment of transfer. Scale bar = 2.5 mm.

(B) Relative root elongation of wild type (Col-0) and ANAC017 overexpressing lines

(ANAC017OE-2 and ANAC017OE-16). Three-day-old seedlings grown on half-strength

MS medium were transferred to medium without (mock) or with 200 nM C17 for two

days. Data represent mean ± SD (n > 10). (C) Root growth of wild type (Col-0),

ANAC017 knockout mutant (anac017-1), cwm2-1 single mutant and cwm2-1 anac017-

1 double mutant in the presence of 200 nM C17. Three-day-old seedlings grown on

half-strength MS medium were transferred to medium with 200 nM C17 for two days.

Arrowheads indicate the root tip position at the moment of transfer. (D-G)

Representative images of root mature zone of wild type (Col-0, D), ANAC017 knockout

mutant (anac017-1, E), cwm2-1 single mutant (F) and cwm2-1 anac017-1 double

mutant (G) in the presence of 200 nM C17. Scale bar = 50µm. (H) Relative root

elongation of wild type (Col-0), ANAC017 knockout mutant (anac017-1), cwm2-1

single mutant and cwm2-1 anac017-1 double mutant in the presence of 200 nM C17.

Three-day-old seedlings grown on half-strength MS medium were transferred to

medium without (mock) or with 200 nM C17 for two days. Data represent mean ± SD

(n > 10). (I) The length of mature cortical cells of wild type (Col-0), ANAC017

knockout mutant (anac017-1), cwm2-1 single mutant and cwm2-1 anac017-1 double

mutant in the presence of 200 nM C17. Data represent mean ± SD (n > 20). Statistically

significant differences are indicated, *P-value < 0.01 (two-sided Student’s t-test).

Parsed CitationsBarkan, A., and Small, I. (2014). Pentatricopeptide repeat proteins in plants. Annu. Rev. Plant Biol. 65, 415-442.

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

Bartoli, C.G., Gómez, F., Martínez, D.E., and Guiamet, J.J. (2004). Mitochondria are the main target for oxidative damage in leavesof wheat (Triticum aestivum L.). J. Exp. Bot. 55, 1663-1669.

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

Beeckman, T., Przemeck, G.K.H., Stamatiou, G., Lau, R., Terryn, N., De Rycke, R., Inzé, D., and Berleth, T. (2002). Geneticcomplexity of cellulose synthase A gene function in Arabidopsis embryogenesis. Plant Physiol. 130, 1883-1893.

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

Bentolila, S., Knight, W., and Hanson, M. (2010). Natural variation in Arabidopsis leads to the identification of REME1, apentatricopeptide repeat-DYW protein controlling the editing of mitochondrial transcripts. Plant Physiol. 154, 1966-1982.

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

Bentolila, S., Oh, J., Hanson, M.R., and Bukowski, R. (2013). Comprehensive high-resolution analysis of the role of an Arabidopsisgene family in RNA editing. PLoS Genet. 9, e1003584.

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

Bleier, L., and Dröse, S. (2013). Superoxide generation by complex III: from mechanistic rationales to functional consequences.Biochim. Biophys. Acta - Bioenerg. 1827, 1320-1331.

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

Boisnard-Lorig, C., Colon-Carmona, A., Bauch, W., Hodge, S., Doerner, P., Bancharel, E., Dumas, C., Haseloff, J., and Berger, F.(2001). Dynamic analyses of the expression of the HISTONE::YFP fusion protein in Arabidopsis show that syncytial endosperm isdivided in mitotic domains. Plant Cell 13, 495-509.

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

Brabham, C., Lei, L., Gu, Y., Stork, J., Barrett, M., and DeBolt, S. (2014). Indaziflam herbicidal action: a potent cellulosebiosynthesis inhibitor. Plant Physiol 166, 1177-1185.

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

Bray, E.A. (2004). Genes commonly regulated by water-deficit stress in Arabidopsis thaliana. J. Exp. Bot. 55, 2331-2341.Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Caño-Delgado, A., Penfield, S., Smith, C., Catley, M., and Bevan, M. (2003). Reduced cellulose synthesis invokes lignification anddefense responses in Arabidopsis thaliana. Plant J. 34, 351-362.

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

Caño-Delgado, A.I., Metzlaff, K., and Bevan, M.W. (2000). The eli1 mutation reveals a link between cell expansion and secondarycell wall formation in Arabidopsis thaliana. Development 127, 3395-3405.

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

Clough, S.J., and Bent, A.F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsisthaliana. Plant J. 16, 735-743.

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

Crowell, E.F., Bischoff, V., Desprez, T., Rolland, A., Stierhof, Y.-D., Schumacher, K., Gonneau, M., Höfte, H., and Vernhettes, S.(2009). Pausing of golgi bodies on microtubules regulates secretion of cellulose synthase complexes in Arabidopsis. Plant Cell 21,1141-1154.

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

De Clercq, I., Vermeirssen, V., Van Aken, O., Vandepoele, K., Murcha, M.W., Law, S.R., Inzé, A., Ng, S., Ivanova, A., Rombaut, D., vande Cotte, B., Jaspers, P., Van de Peer, Y., Kangasjärvi, J., Whelan, J., and Van Breusegem, F. (2013). The membrane-bound NACtranscription factor ANAC013 functions in mitochondrial retrograde regulation of the oxidative stress response in Arabidopsis.Plant Cell 25, 3472-3490.

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

de Longevialle, A.F., Meyer, E.H., Andres, C., Taylor, N.L., Lurin, C., Millar, A.H., and Small, I.D. (2007). The pentatricopeptide repeatgene OTP43 is required for trans-splicing of the mitochondrial nad1 Intron 1 in Arabidopsis thaliana. Plant Cell 19, 3256-3265.

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

Deng, Y., Srivastava, R., and Howell, S.H. (2013). Endoplasmic reticulum (ER) stress response and its physiological roles in plants.Int. J. Mol. Sci. 14, 8188-8212.

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

Desprez, T., Vernhettes, S., Fagard, M., Refrégier, G., Desnos, T., Aletti, E., Py, N., Pelletier, S., and Höfte, H. (2002). Resistanceagainst herbicide isoxaben and cellulose deficiency caused by distinct mutations in same cellulose synthase isoform CESA6. PlantPhysiol. 128, 482-490.

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

Desprez, T., Juraniec, M., Crowell, E.F., Jouy, H., Pochylova, Z., Parcy, F., Höfte, H., Gonneau, M., and Vernhettes, S. (2007).Organization of cellulose synthase complexes involved in primary cell wall synthesis in Arabidopsis thaliana. Proc. Natl. Acad. Sci.USA 104, 15572-15577.

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

Duncan, O., Taylor, N.L., Carrie, C., Eubel, H., Kubiszewski-Jakubiak, S., Zhang, B., Narsai, R., Millar, A.H., and Whelan, J. (2011).Multiple lines of evidence localize signaling, morphology, and lipid biosynthesis machinery to the mitochondrial outer membraneof Arabidopsis. Plant Physiol. 157, 1093-1113.

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

Ellis, C., and Turner, J.G. (2001). The Arabidopsis mutant cev1 has constitutively active jasmonate and ethylene signal pathwaysand enhanced resistance to pathogens. Plant Cell 13, 1025-1033.

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

Ellis, C., Karafyllidis, I., Wasternack, C., and Turner, J.G. (2002). The Arabidopsis mutant cev1 links cell wall signaling to jasmonateand ethylene responses. Plant Cell 14, 1557-1566.

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

Elthon, T.E., Nickels, R.L., and McIntosh, L. (1989). Monoclonal antibodies to the alternative oxidase of higher plant mitochondria.Plant Physiol. 89, 1311-1317.

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

Endler, A., and Persson, S. (2011). Cellulose synthases and synthesis in Arabidopsis. Mol. Plant 4, 199-211.Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Eubel, H., Braun, H.-P., and Millar, A.H. (2005). Blue-native PAGE in plants: a tool in analysis of protein-protein interactions. PlantMethods 1, 11.

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

Francs-Small, C.C.d., Kroeger, T., Zmudjak, M., Ostersetzer-Biran, O., Rahimi, N., Small, I., and Barkan, A. (2012). A PORR domainprotein required for rpl2 and ccmFC intron splicing and for the biogenesis of c-type cytochromes in Arabidopsis mitochondria.Plant J. 69, 996-1005.

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

Fujita, M., Fujita, Y., Noutoshi, Y., Takahashi, F., Narusaka, Y., Yamaguchi-Shinozaki, K., and Shinozaki, K. (2006). Crosstalkbetween abiotic and biotic stress responses: a current view from the points of convergence in the stress signaling networks.Curr. Opin. Plant Biol. 9, 436-442.

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

Gechev, T.S., Van Breusegem, F., Stone, J.M., Denev, I., and Laloi, C. (2006). Reactive oxygen species as signals that modulateplant stress responses and programmed cell death. BioEssays 28, 1091-1101.

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

Giraud, E., Van Aken, O., Ho, L.H.M., and Whelan, J. (2009). The transcription factor ABI4 is a regulator of mitochondrial retrogradeexpression of ALTERNATIVE OXIDASE1a. Plant Physiol. 150, 1286-1296.

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

Giraud, E., Ho, L.H.M., Clifton, R., Carroll, A., Estavillo, G., Tan, Y.-F., Howell, K.A., Ivanova, A., Pogson, B.J., Millar, A.H., andWhelan, J. (2008). The absence of ALTERNATIVE OXIDASE1a in Arabidopsis results in acute sensitivity to combined light anddrought stress. Plant Physiol. 147, 595-610.

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

Gutierrez, R., Lindeboom, J.J., Paredez, A.R., Emons, A.M.C., and Ehrhardt, D.W. (2009). Arabidopsis cortical microtubules positioncellulose synthase delivery to the plasma membrane and interact with cellulose synthase trafficking compartments. Nat. Cell Biol.11, 797-806.

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

Hamann, T. (2015). The plant cell wall integrity maintenance mechanism—Concepts for organization and mode of action. Plant CellPhysiol. 56, 215-223.

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

Harholt, J., Jensen, J.K., Sorensen, S.O., Orfila, C., Pauly, M., and Scheller, H.V. (2006). ARABINAN DEFICIENT 1 is a putativearabinosyltransferase involved in biosynthesis of pectic arabinan in Arabidopsis. Plant Physiol. 140, 49-58.

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

He, J., Duan, Y., Hua, D., Fan, G., Wang, L., Liu, Y., Chen, Z., Han, L., Qu, L.J., and Gong, Z. (2012). DEXH box RNA helicase-mediated mitochondrial reactive oxygen species production in Arabidopsis mediates crosstalk between abscisic acid and auxinsignaling. Plant Cell 24, 1815-1833.

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

Hématy, K., Sado, P.-E., Van Tuinen, A., Rochange, S., Desnos, T., Balzergue, S., Pelletier, S., Renou, J.-P., and Höfte, H. (2007). Areceptor-like kinase mediates the response of Arabidopsis cells to the inhibition of cellulose synthesis. Curr. Biol. 17, 922-931.

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

Howell, S.H. (2013). Endoplasmic reticulum stress responses in plants. Annu. Rev. Plant Biol. 64, 477-499.Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Huang, S., Van Aken, O., Schwarzlander, M., Belt, K., and Millar, A.H. (2016). Roles of mitochondrial reactive oxygen species incellular signalling and stress response in plants. Plant Physiol.

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

Iraki, N.M., Bressan, R.A., Hasegawa, P.M., and Carpita, N.C. (1989). Alteration of the physical and chemical structure of the primarycell wall of growth-limited plant cells adapted to osmotic stress. Plant Physiol. 91, 39-47.

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

Karimi, M., Inzé, D., and Depicker, A. (2002). GATEWAYTM vectors for Agrobacterium-mediated plant transformation. Trends PlantSci. 7, 193-195.

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

Koprivova, A., des Francs-Small, C.C., Calder, G., Mugford, S.T., Tanz, S., Lee, B.R., Zechmann, B., Small, I., and Kopriva, S. (2010).Identification of a pentatricopeptide repeat protein implicated in splicing of intron 1 of mitochondrial nad7 transcripts. The Journal

of biological chemistry 285, 32192-32199.Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Kranz, R.G., Richard-Fogal, C., Taylor, J.-S., and Frawley, E.R. (2009). Cytochrome c biogenesis: mechanisms for covalentmodifications and trafficking of heme and for heme-iron redox control. Microbiol. Mol. Biol. Rev. 73, 510-528.

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

Lei, L., Singh, A., Bashline, L., Li, S., Yingling, Y.G., and Gu, Y. (2015). CELLULOSE SYNTHASE INTERACTIVE1 is required for fastrecycling of cellulose synthase complexes to the plasma membrane in Arabidopsis. Plant Cell 27, 2926-2940.

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

Liu, Y., He, J., Chen, Z., Ren, X., Hong, X., and Gong, Z. (2010). ABA overly-sensitive 5 (ABO5), encoding a pentatricopeptide repeatprotein required for cis-splicing of mitochondrial nad2 intron 3, is involved in the abscisic acid response in Arabidopsis. Plant J 63,749-765.

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

Mavridou, D.A.I., Ferguson, S.J., and Stevens, J.M. (2013). Cytochrome c assembly. IUBMB Life 65, 209-216.Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

May, M.J., and Leaver, C.J. (1993). Oxidative stimulation of glutathione synthesis in Arabidopsis thaliana suspension cultures.Plant Physiol. 103, 621-627.

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

McFarlane, H.E., Döring, A., and Persson, S. (2014). The cell biology of cellulose synthesis. Annu. Rev. Plant Biol. 65, 69-94.Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Meyer, E.H., Tomaz, T., Carroll, A.J., Estavillo, G., Delannoy, E., Tanz, S.K., Small, I.D., Pogson, B.J., and Millar, A.H. (2009).Remodeled respiration in ndufs4 with low phosphorylation efficiency suppresses Arabidopsis germination and growth and alterscontrol of metabolism at night. Plant Physiol. 151, 603-619.

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

Miart, F., Desprez, T., Biot, E., Morin, H., Belcram, K., Höfte, H., Gonneau, M., and Vernhettes, S. (2014). Spatio-temporal analysis ofcellulose synthesis during cell plate formation in Arabidopsis. Plant J. 77, 71-84.

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

Millar, A.H., Sweetlove, L.J., Giege, P., and Leaver, C.J. (2001). Analysis of the Arabidopsis mitochondrial proteome. Plant Physiol127, 1711-1727.

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

Millar, A.H., Whelan, J., Soole, K.L., and Day, D.A. (2011). Organization and regulation of mitochondrial respiration in plants. Annu.Rev. Plant Biol. 62, 79-104.

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

Murashige, T., and Skoog, F. (1962). A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant.15, 473-497.

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

Murcha, M.W., and Whelan, J. (2015). Isolation of intact mitochondria from the model plant species Arabidopsis thaliana and Oryzasativa. Methods Mol. Biol. 1305, 1-12.

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

Ng, S., De Clercq, I., Van Aken, O., Law, S.R., Ivanova, A., Willems, P., Giraud, E., Van Breusegem, F., and Whelan, J. (2014).Anterograde and retrograde regulation of nuclear genes encoding mitochondrial proteins during growth, development, andstress. Mol. Plant 7, 1075-1093.

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

Ng, S., Ivanova, A., Duncan, O., Law, S.R., Van Aken, O., De Clercq, I., Wang, Y., Carrie, C., Xu, L., Kmiec, B., Walker, H., VanBreusegem, F., Whelan, J., and Giraud, E. (2013a). A membrane-bound NAC transcription factor, ANAC017, mediates mitochondrialretrograde signaling in Arabidopsis. Plant Cell 25, 3450-3471.

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

Ng, S., Giraud, E., Duncan, O., Law, S.R., Wang, Y., Xu, L., Narsai, R., Carrie, C., Walker, H., Day, D.A., Blanco, N.E., Strand, Å.,Whelan, J., and Ivanova, A. (2013b). Cyclin-dependent kinase E1 (CDKE1) provides a cellular switch in plants between growth andstress responses. J. Biol. Chem. 288, 3449-3459.

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

Persson, S., Paredez, A., Carroll, A., Palsdottir, H., Doblin, M., Poindexter, P., Khitrov, N., Auer, M., and Somerville, C.R. (2007).Genetic evidence for three unique components in primary cell-wall cellulose synthase complexes in Arabidopsis. Proc. Natl. Acad.Sci. USA 104, 15566-15571.

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

Piro, G., Leucci, M.R., Waldron, K., and Dalessandro, G. (2003). Exposure to water stress causes changes in the biosynthesis ofcell wall polysaccharides in roots of wheat cultivars varying in drought tolerance. Plant Sci. 165, 559-569.

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

Rhoads, D.M., and Subbaiah, C.C. (2007). Mitochondrial retrograde regulation in plants. Mitochondrion 7, 177-194.Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Rhoads, D.M., Umbach, A.L., Subbaiah, C.C., and Siedow, J.N. (2006). Mitochondrial reactive oxygen species. Contribution tooxidative stress and interorganellar signaling. Plant Physiol. 141, 357-366.

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

Schmitz-Linneweber, C., and Small, I. (2008). Pentatricopeptide repeat proteins: a socket set for organelle gene expression.Trends Plant Sci. 13, 663-670.

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

Skirycz, A., De Bodt, S., Obata, T., De Clercq, I., Claeys, H., De Rycke, R., Andriankaja, M., Van Aken, O., Van Breusegem, F., Fernie,A.R., and Inze, D. (2010). Developmental stage specificity and the role of mitochondrial metabolism in the response of Arabidopsisleaves to prolonged mild osmotic stress. Plant Physiol 152, 226-244.

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

Srivastava, R., Deng, Y., Shah, S., Rao, A.G., and Howell, S.H. (2013). BINDING PROTEIN is a master regulator of the endoplasmicreticulum stress sensor/transducer bZIP28 in Arabidopsis. Plant Cell 25, 1416-1429.

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

Starkov, A.A. (2008). The role of mitochondria in reactive oxygen species metabolism and signaling. Ann. NY Acad.Sci. 1147, 37-52.Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Sung, T.-Y., Tseng, C.-C., and Hsieh, M.-H. (2010). The SLO1 PPR protein is required for RNA editing at multiple sites with similarupstream sequences in Arabidopsis mitochondria. Plant J. 63, 499-511.

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

Sweet, W.J., Morrison, J.C., Labavitch, J.M., and Matthews, M.A. (1990). Altered synthesis and composition of cell wall of grape(Vitis vinifera L.) leaves during expansion and growth-inhibiting water deficits. Plant Cell Physiol. 31, 407-414.

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

Takenaka, M. (2010). MEF9, an E-subclass pentatricopeptide repeat protein, is required for an RNA editing event in the nad7transcript in mitochondria of Arabidopsis. Plant Physiol. 152, 939-947.

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

Takenaka, M., Verbitskiy, D., Zehrmann, A., and Brennicke, A. (2010). Reverse genetic screening identifies five E-class PPRproteins involved in RNA editing in mitochondria of Arabidopsis thaliana. J. Biol. Chem. 285, 27122-27129.

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

Tenhaken, R. (2015). Cell wall remodeling under abiotic stress. Front Plant Sci 5, 771.Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Updegraff, D.M. (1969). Semimicro determination of cellulose in biological materials. Anal. Biochem. 32, 420-424.Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Van Aken, O., Zhang, B., Law, S., Narsai, R., and Whelan, J. (2013). AtWRKY40 and AtWRKY63 modulate the expression of stress-responsive nuclear genes encoding mitochondrial and chloroplast proteins. Plant Physiol. 162, 254-271.

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

Van Aken, O., De Clercq, I., Ivanova, A., Law, S.R., Van Breusegem, F., Millar, A.H., and Whelan, J. (2016). Mitochondrial andchloroplast stress responses are modulated in distinct touch and chemical inhibition phases in Arabidopsis. Plant Physiol. ?dx.?doi.?org/?10.?1104/?pp.?16.?00273.

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

Vandavasi, V.G., Putnam, D.K., Zhang, Q., Petridis, L., Heller, W.T., Nixon, B.T., Haigler, C.H., Kalluri, U., Coates, L., Langan, P.,Smith, J.C., Meiler, J., and O'Neill, H. (2016). A Structural Study of CESA1 Catalytic Domain of Arabidopsis Cellulose SynthesisComplex: Evidence for CESA Trimers. Plant Physiol 170, 123-135.

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

Vanderauwera, S., Vandenbroucke, K., Inze, A., van de Cotte, B., Muhlenbock, P., De Rycke, R., Naouar, N., Van Gaever, T., VanMontagu, M.C., and Van Breusegem, F. (2012). AtWRKY15 perturbation abolishes the mitochondrial stress response that steersosmotic stress tolerance in Arabidopsis. Proc Natl Acad Sci U S A 109, 20113-20118.

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

Vanlerberghe, G.C. (2013). Alternative oxidase: a mitochondrial respiratory pathway to maintain metabolic and signalinghomeostasis during abiotic and biotic stress in plants. Int. J. Mol. Sci. 14, 6805-6847.

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

Verbitskiy, D., Zehrmann, A., van der Merwe, J.A., Brennicke, A., and Takenaka, M. (2010). The PPR protein encoded by theLOVASTATIN INSENSITIVE 1 gene is involved in RNA editing at three sites in mitochondria of Arabidopsis thaliana. Plant J. 61,446-455.

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

Wang, Y., Carrie, C., Giraud, E., Elhafez, D., Narsai, R., Duncan, O., Whelan, J., and Murcha, M.W. (2012). Dual location of themitochondrial preprotein transporters B14.7 and Tim23-2 in complex I and the TIM17:23 complex in Arabidopsis links mitochondrialactivity and biogenesis. Plant Cell 24, 2675-2695.

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

Welchen, E., Hildebrandt, T.M., Lewejohann, D., Gonzalez, D.H., and Braun, H.-P. (2012). Lack of cytochrome c in Arabidopsisdecreases stability of Complex IV and modifies redox metabolism without affecting Complexes I and III. Biochim. Biophys. Acta -Bioenerg. 1817, 990-1001.

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

Wolf, S., Hématy, K., and Höfte, H. (2012). Growth control and cell wall signaling in plants. Annu. Rev. Plant Biol. 63, 381-407.Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Wu, J., Sun, Y., Zhao, Y., Zhang, J., Luo, L., Li, M., Wang, J., Yu, H., Liu, G., Yang, L., Xiong, G., Zhou, J.-M., Zuo, J., Wang, Y., and Li,

J. (2015). Deficient plastidic fatty acid synthesis triggers cell death by modulating mitochondrial reactive oxygen species. Cell Res.25, 621-633.

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

Xia, Y., Lei, L., Brabham, C., Stork, J., Strickland, J., Ladak, A., Gu, Y., Wallace, I., and DeBolt, S. (2014). Acetobixan, an inhibitor ofcellulose synthesis identified by microbial bioprospecting. PLoS ONE 9, e95245.

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

Yang, L., Zhang, J., He, J., Qin, Y., Hua, D., Duan, Y., Chen, Z., and Gong, Z. (2014). ABA-mediated ROS in mitochondria regulateroot meristem activity by controlling PLETHORA expression in Arabidopsis. PLoS Genet 10, e1004791.

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

Zehrmann, A., Verbitskiy, D., van der Merwe, J.A., Brennicke, A., and Takenaka, M. (2009). A DYW domain-containingpentatricopeptide repeat protein is required for RNA editing at multiple sites in mitochondria of Arabidopsis thaliana. Plant Cell 21,558-567.

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

Zhu, J., Lee, B.H., Dellinger, M., Cui, X., Zhang, C., Wu, S., Nothnagel, E.A., and Zhu, J.K. (2010). A cellulose synthase-like protein isrequired for osmotic stress tolerance in Arabidopsis. Plant J 63, 128-140.

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

Zhu, Q., Dugardeyn, J., Zhang, C., Muhlenbock, P., Eastmond, P.J., Valcke, R., De Coninck, B., Oden, S., Karampelias, M., Cammue,B.P., Prinsen, E., and Van Der Straeten, D. (2014). The Arabidopsis thaliana RNA editing factor SLO2, which affects themitochondrial electron transport chain, participates in multiple stress and hormone responses. Mol Plant 7, 290-310.

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

Zsigmond, L., Rigo, G., Szarka, A., Szekely, G., Otvos, K., Darula, Z., Medzihradszky, K.F., Koncz, C., Koncz, Z., and Szabados, L.(2008). Arabidopsis PPR40 connects abiotic stress responses to mitochondrial electron transport. Plant Physiol 146, 1721-1737.

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