Plant Physiology Preview. Published on July 1, 2015, as DOI:10.1104/pp.15.00609

1

Running title: WDL5 in Ethylene-Regulated Hypocotyl Elongation

2 3

Corresponding author

4

Tonglin Mao

5

State Key Laboratory of Plant Physiology and Biochemistry, Department of Plant

6

Sciences, College of Biological Sciences, China Agricultural University, Beijing

7

100193, China. Telephone: +8610-62732330. FAX: +8610-62732330. E-mail:

8

[email protected]

9 10 11 12 13 14 15 16 17 18

Number of characters in the manuscript (include spaces): 64328

19

Number of 1-column figures: 10

20

Number of 2-column figures: 6

21 22 23 24 25 26 27 28 29

Keywords: WDL5, Ethylene, Cortical microtubule, Hypocotyl elongation,

30

Arabidopsis 1 Downloaded from www.plantphysiol.org on July 9, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

Copyright 2015 by the American Society of Plant Biologists

31

Ethylene Regulates Arabidopsis Microtubule-Associated Protein WDL5 in

32

Etiolated Hypocotyl Elongation

33

Jingbo Sun1, Qianqian Ma1, and Tonglin Mao*

34 35

State Key Laboratory of Plant Physiology and Biochemistry; Department of Plant

36

Sciences, College of Biological Sciences, China Agricultural University, Beijing

37

100193, China

38 39 40

1

41

*to whom correspondence should be addressed. E-mail: [email protected];

42

Fax: +8610-62732330

These authors contributed equally to this work.

43 44 45 46

The author responsible for the distribution of materials integral to the findings

47

presented in this article in accordance with the policy described in the Instructions for

48

Authors (www.plantphysiol.org) is Tonglin Mao ([email protected]).

49 50 51 52 53 54 55 56

Synopsis

57

This study reveals a mechanism wherein the plant hormone ethylene controls

58

expression of the microtubule-associated protein WDL5 and regulates cortical

59

microtubule organization to mediate etiolated hypocotyl cell elongation in

60

Arabidopsis. 2 Downloaded from www.plantphysiol.org on July 9, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

61

Footnotes:

62

This research was supported by grants from the National Basic Research Program of

63

China (2012CB114200 to T.M.), the Natural Science Foundation of China

64

(31471272 and 31222007 to T. M.), and Program for New Century Excellent Talents

65

in University (NCET-12-0523 to T.M.).

66 67

*Address correspondence to [email protected]

68

3 Downloaded from www.plantphysiol.org on July 9, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

69

ABSTRACT

70

The phytohormone ethylene plays crucial roles in the negative regulation of plant

71

etiolated hypocotyl elongation. The microtubule cytoskeleton also participates in

72

hypocotyl cell growth. However, it remains unclear if ethylene signaling-mediated

73

etiolated hypocotyl elongation involves the microtubule cytoskeleton. In the present

74

study, we functionally identified the previously uncharacterized

75

microtubule-associated protein WAVE-DAMPENED2-LIKE5 (WDL5) as a

76

microtubule stabilizing protein that plays a positive role in ethylene-regulated

77

etiolated hypocotyl cell elongation. ETHYLENE-INSENSITIVE 3 (EIN3), a key

78

transcription factor in the ethylene signaling pathway, directly targets and upregulates

79

WDL5. Etiolated hypocotyls from a WDL5 loss-of-function mutant (wdl5-1) were

80

more insensitive to ACC treatment than the wildtype. Decreasing WDL5 expression

81

partially rescued the shorter-etiolated-hypocotyl phenotype in the ethylene

82

overproduction mutant eto1-1. Reorganization of cortical microtubules in etiolated

83

hypocotyl cells from the wdl5-1 mutant was less sensitive to ACC treatment. These

84

findings indicate that WDL5 is an important participant in ethylene signaling

85

inhibition of etiolated hypocotyl growth. This study reveals a mechanism involved in

86

ethylene regulation of microtubules through WDL5 to inhibit etiolated hypocotyl cell

87

elongation.

88

4 Downloaded from www.plantphysiol.org on July 9, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

89

INTRODUCTION

90

Skotomorphogenesis occurs as buried seedlings fully elongate their hypocotyls

91

upward in search of the soil surface. When elongated hypocotyls encounter

92

mechanical obstacles during seedling extrusion from the soil, inhibition of rapid

93

etiolated hypocotyl elongation is required to optimize the seedling’s ability to push

94

through the soil without damaging its shoot meristem. Disturbing this physiological

95

process significantly affects seedling emergence from the soil and survival (Zhong et

96

al., 2014). The phytohormone ethylene plays a crucial role in the negative regulation

97

of hypocotyl elongation in the dark. Ethylene functions through five membrane-bound

98

receptors (ETR1, ERS1, ETR2, ERS2, and EIN4) and a well-defined signal

99

transduction pathway to activate the redundant nuclear-localized transcription factors

100

ETHYLENE-INSENSITIVE 3 (EIN3) and EIN3-like 1 (EIL1). EIN3 and EIL1

101

specifically bind to the promoters of ethylene response target genes to activate or

102

repress their expression, thereby modulating ethylene-related responses in plants

103

(Boutrot et al., 2010; Zhang et al., 2011; Chang et al., 2013). Abundance of the EIN3

104

protein rapidly increases with ethylene treatment, but is targeted by SCFEBF1/EBF2

105

complexes and degraded in the absence of ethylene (Guo and Ecker, 2003; Potuschak

106

et al., 2003). One of the most widely documented ethylene responses in etiolated

107

seedlings is the triple response, including a short, thickened hypocotyl when

108

dark-grown Arabidopsis thaliana seedlings are treated with ethylene or its

109

biosynthetic precursor 1-aminocyclopropane-1-carboxylic acid (ACC) (Bleecker et al.,

110

1988; Ecker, 1995). Ethylene and ACC stimulate hypocotyl elongation in the light but

111

suppress etiolated hypocotyl elongation in the dark, largely due to concomitant

112

activation of two contrasting pathways (Ecker, 1995; Zhong et al., 2012). Genetic

113

evidence has shown that ethylene-overproduced or constitutive-ethylene-response

114

mutants generally display defective etiolated hypocotyl cell growth phenotypes. For

115

example, the ethylene-overproducing mutant eto1-1 and the constitutive ethylene

116

response CTR1 (constitutive triple response1) mutant ctr1-1 have shorter etiolated

117

hypocotyls than wild-type seedlings (WT) in the dark (Kieber et al., 1993). Treatment

118

with ACC obviously inhibited etiolated hypocotyl elongation of wild-type seedlings, 5 Downloaded from www.plantphysiol.org on July 9, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

119

but not ein3eil1 seedlings, and overexpression of EIN3 significantly inhibited

120

hypocotyl elongation in the dark (An et al., 2010), demonstrating that EIN3 and EIL1

121

are required for ethylene-inhibited hypocotyl elongation in the dark. Although

122

ethylene has been implicated in the regulation of hypocotyl growth in the dark, the

123

molecular mechanisms regarding EIN3 regulation of downstream effectors that may

124

directly participate in inhibiting etiolated hypocotyl elongation are largely unknown.

125

Cortical microtubules orient cellulose fibrils to control plant cell growth by

126

building the mechanical properties of the cell wall (Lloyd and Chan, 2008; Lloyd,

127

2011; Bashline et al., 2014; Lei et al., 2014). Multiple approaches have demonstrated

128

that regulation of the stabilization, organization and dynamics of cortical microtubules

129

is pivotal for hypocotyl cell growth. Etiolated Arabidopsis seedlings exhibit stunted

130

hypocotyls when the microtubule-disrupting drug propyzamide is used to disturb

131

cortical microtubules (Le et al., 2005). Mutation or overexpression of many

132

microtubule-associated proteins (MAPs) also results in abnormal etiolated hypocotyl

133

cell elongation by altering the stability and organization of cortical microtubules. For

134

example, overexpression of the microtubule plus-end tracking protein SPIRAL1

135

promotes etiolated hypocotyl elongation by stabilizing cortical microtubules, whereas

136

overexpression of MICROTUBULE-DESTABILIZING PROTEIN25 (MDP25)

137

inhibits etiolated hypocotyl elongation by destabilizing cortical microtubules

138

(Nakajima et al., 2004, 2006; Li et al., 2011; Galva et al., 2014).

139

Hypocotyl elongation is strongly influenced by developmental and environmental

140

cues. Studies have detailed the mechanisms involved in hypocotyl cell elongation that

141

are regulated by light, phytohormones, and transcription factors (Niwa et al., 2009；

142

Luo et al., 2010; Fan et al., 2012). However, the role of microtubules in these

143

physiological processes remains to be determined. A recent study showed that

144

Arabidopsis MICROTUBULE-DESTABILIZING PROTEIN40 (MDP40) is involved

145

in brassinosteroid (BR) signaling promotion of hypocotyl growth (Wang et al., 2012).

146

Although ethylene has been reported to affect the organization of cortical

147

microtubules in plant cells (Takahashi et al., 2003; Le et al., 2005; Soga et al., 2010;

148

Polko et al., 2012), molecular mechanisms regarding the effects of ethylene signaling 6 Downloaded from www.plantphysiol.org on July 9, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

149

on microtubule regulation in mediating hypocotyl elongation are largely unclear.

150

Identification of microtubule-associated proteins involved in ethylene-mediated

151

hypocotyl elongation will facilitate the understanding of underlying mechanisms of

152

ethylene-regulated cell growth.

153

WDL5 belongs to the microtubule-associated protein WAVE-DAMPENED 2

154

(WVD2)/WVD2-LIKE (WDL) family in Arabidopsis (Yuen et al., 2003; Perrin et al.,

155

2007). Seedlings with constitutive WVD2 expression exhibit short, thick stems and

156

roots and inverted handedness of twisting hypocotyls and roots (Yuen et al., 2003).

157

WDL3 is a negative regulator of hypocotyl elongation in the light and is degraded by

158

the ubiquitin-26S proteasome-dependent pathway in the dark (Liu et al., 2013),

159

suggesting diverse physiological roles of WVD2/WDL proteins in plant growth and

160

plant cell morphogenesis. In this study, we demonstrated that ethylene regulates

161

microtubules through WDL5, which is targeted by EIN3 and upregulated by ethylene,

162

to inhibit etiolated hypocotyl cell elongation. This study demonstrates that WDL5 is

163

involved in ethylene-mediated etiolated hypocotyl cell elongation by altering the

164

organization and stability of cortical microtubules.

165 166 167 168

RESULTS

169

WDL5 Is an Ethylene-Upregulated Gene

170

Given that WDL5 expression was shown to be regulated by ethylene in a

171

microarray assay and its homolog WDL3 is involved in hypocotyl elongation in

172

Arabidopsis (Zhong et al., 2009; Liu et al., 2013), we speculated that WDL5 may

173

play a role in ethylene-regulated hypocotyl cell elongation. We first determined

174

whether and how ethylene regulates WDL5 expression.

175

RNA was purified from etiolated seedlings of the ethylene overproduction mutant

176

eto1-1 and ethylene insensitive mutant ein2-5 and quantitative real-time PCR

177

analyses were performed. WDL5 expression was much higher in the eto1-1 mutant,

178

but lower in the ein2-5 mutant compared to the wildtype (Fig. 1A). After etiolated 7 Downloaded from www.plantphysiol.org on July 9, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

179

wild-type seedlings were treated with 100 μM ACC, quantitative real-time PCR

180

showed that WDL5 expression was induced by ACC treatment, with peak levels

181

detected 6 h after treatment (Fig. 1B). These results indicate that ethylene

182

upregulates WDL5 expression.

183 184

WDL5 Functions as a Positive Regulator in Ethylene-Mediated Etiolated

185

Hypocotyl Cell Elongation

186

WDL5 expression is significantly upregulated by ethylene, suggesting a potential

187

role of WDL5 in ethylene-regulated hypocotyl cell elongation. To determine the

188

function of WDL5, the T-DNA insertion mutant wdl5-1 was obtained from TAIR

189

(CS436432). The homozygous wdl5-1 mutant contained a T-DNA insertion in the

190

intron, and a full-length transcript was not detected by reverse transcription

191

polymerase chain reaction (RT-PCR) (Fig. 2A and 2B). However, a partial transcript

192

upstream of the T-DNA insertion site was identified (Supplemental Fig. S1). The

193

wdl5-1 phenotype indicated that the function of WDL5 was abolished or

194

dramatically affected in the mutant (Fig. 2C and 2D). In addition, another WDL5

195

T-DNA insertion allele (wdl5-2-CS434701) with a T-DNA insertion site in the exon

196

was detected, although a full-length WDL5 transcript was not detected. This mutant

197

exhibited a phenotype similar to wdl5-1 (Supplemental Fig. S2A-C).

198

To analyze the role of WDL5 in ethylene-mediated etiolated hypocotyl elongation,

199

wild-type and wdl5-1 seedlings were cultured on MS medium containing various

200

concentrations of ACC, and etiolated hypocotyl lengths were measured. Observation

201

of 5-day-old dark-grown wdl5-1 seedlings revealed that the hypocotyl length was

202

longer than in wild-type plants without ACC treatment. This phenotype was

203

complemented by PWDL5:WDL5 (Supplemental Fig. S3A-C), indicating that the

204

aberrant etiolated hypocotyl phenotype in wdl5-1 is associated with WDL5

205

expression levels. Hypocotyl length in 5-day-old etiolated seedlings from the

206

wildtype was dramatically reduced in the presence of ACC, while etiolated

207

hypocotyls were much longer in wdl5-1 seedlings grown on the same medium (Fig.

208

2C). The effects of ACC on hypocotyl elongation were more pronounced in the 8 Downloaded from www.plantphysiol.org on July 9, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

209

wildtype and decreased in the wdl5-1 mutant at all concentrations (Fig. 2D),

210

indicating that wdl5-1 mutant seedlings are much less sensitive to ACC in etiolated

211

hypocotyl elongation than the wildtype. Thus, these observations demonstrate that

212

WDL5 plays a positive role in ethylene-regulated etiolated hypocotyl elongation.

213

Scanning electronic microscopy revealed that the length of etiolated hypocotyl

214

cells was longer in wdl5-1 than in wild-type seedlings in the presence of 10 μM ACC,

215

particularly in the middle and top regions (Fig. 2E). Paired Student’s t-test indicated

216

that the difference in relative cell lengths between the wildtype and wdl5-1 mutant in

217

response to ACC was significant (Fig. 2F). The number of cells in individual

218

hypocotyl-epidermal cell files in wild-type and wdl5-1 seedlings was similar

219

(approximately 20 to 22). These results suggest that WDL5 plays a positive role in

220

ethylene inhibition of etiolated hypocotyl cell elongation.

221 222

WDL5 Is an EIN3 Target Gene

223

Redundant transcription factors EIN3 and EIL1 play central roles in ethylene

224

regulation of plant growth and development. EIN3 and EIL1 bind to EIN3-binding

225

sites (EBSs) on target gene promoters (Kosugi and Ohashi, 2000; Zhong et al., 2009;

226

Shi et al., 2012). To determine if ethylene signaling directly regulates WDL5

227

expression, we analyzed the WDL5 promoter sequence. Bioinformatics’ analysis has

228

revealed that the WDL5 promoter regions contain three putative EBSs (Zhong et al.,

229

2009) (located at -389 – -393, -1111 – -1115, and -1205 – -1209 upstream of the

230

transcription start site) (Fig. 3A). To determine whether the EIN3 protein binds to the

231

WDL5 promoter, chromatin immunoprecipitation (ChIP) was performed. An

232

EIN3-3×FLAG fusion protein was expressed using an estradiol-inducible promoter

233

(Chen et al., 2009) and immunoprecipitated using an antibody recognizing the

234

FLAG tag. Genomic DNA fragments that co-immunoprecipitated with

235

EIN3-3×FLAG were analyzed using quantitative real-time PCR. Chromatin

236

immunoprecipitated with the anti-FLAG antibody was enriched in fragments P1

237

(located from -332 to -510 upstream of the transcription start site) and P2

238

(containing two close EBSs, located from -1086 to -1247 upstream of the 9 Downloaded from www.plantphysiol.org on July 9, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

239

transcription start site) in WDL5 promoter, but not in a control in which DNA

240

precipitated without the anti-FLAG antibody (Fig. 3B).

241

We further tested direct binding of EIN3 to P1 and P2 of the WDL5 promoter with

242

electrophoretic mobility shift assays (EMSAs) using NHS-biotin-labeled DNA

243

fragments of the WDL5 promoter and a bacterially expressed truncated GST-EIN3

244

protein (amino acids 141 to 352) containing the DNA binding domain in vitro (Chen

245

et al., 2009). The results showed that the GST-EIN3 fusion protein bound to P1 and

246

P2, but not the -536 to -690 region (P3, without putative EBSs, upstream of the

247

transcription start site) of the WDL5 promoter. Moreover, binding was abolished by

248

addition of increasing amounts of unlabeled P1 and P2 probes (Fig. 3C and 3D),

249

indicating that EIN3 can directly bind to the WDL5 promoter in vitro. These results

250

demonstrate that WDL5 is an EIN3 target gene. A previous study showed that

251

ethylene constantly activates a hypocotyl elongation-inhibiting pathway mediated by

252

AP2-type transcription factor ethylene response factor 1 (ERF1) in the dark (Zhong

253

et al., 2012). We evaluated the WDL5 promoter sequence and found a typical

254

ERF1-binding motif with a variant base site (AGCCGCT) (Supplemental Fig. S4A,

255

see asterisk). A previous study demonstrated that this site is crucial for DNA binding

256

of ERF1 (Fujimoto et al., 2000). EMSA in the present study also showed that ERF1

257

did not bind to the WDL5 promoter due to this variant site (Supplemental Fig. S4B),

258

demonstrating that ethylene regulates WDL5 expression through EIN3, but not

259

ERF1.

260 261

Decreasing WDL5 Expression Partially Suppresses Short Etiolated Hypocotyls

262

in the eto1-1 Mutant

263

Because the above-mentioned results show that WDL5 is an EIN3-target and an

264

ethylene-upregulated gene, we hypothesized that decreased WDL5 expression could

265

suppress the short etiolated hypocotyl phenotype induced by overproduction of

266

ethylene. We crossed wdl5-1 with eto1-1 to create a wdl5-1eto1-1 double mutant. All

267

15 of the wdl5-1eto1-1 lines obtained exhibited the longer-etiolated-hypocotyl

268

phenotype, and line 2 was selected for further analyses (Fig. 4B; data not shown). 10 Downloaded from www.plantphysiol.org on July 9, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

269

RT-PCR showed that WDL5 transcription levels were considerably decreased in

270

wdl5-1eto1-1 seedlings (Fig. 4A). Decreased WDL5 expression was correlated with a

271

dramatic increase in the etiolated hypocotyl length of eto1-1 mutants in 5-day-old

272

etiolated seedlings (Fig. 4B and 4C). Scanning electronic microscopy revealed that

273

etiolated hypocotyl cell length in eto1-1 mutants was increased when WDL5

274

expression was reduced (Fig. 4D). Hypocotyl cell length was significantly increased

275

in wdl5-1eto1-1 mutants (Fig. 4E). This evidence demonstrates that WDL5 is a

276

downstream factor in the ethylene signaling pathway and is associated with inhibited

277

etiolated hypocotyl cell elongation in response to ethylene.

278 279

WDL5 Regulates Cortical Microtubule Organization and Stability in Response

280

to Ethylene

281

Because cortical microtubule organization is associated with the growth status of

282

etiolated hypocotyls (Le et al., 2005; Crowell et al., 2011) and WDL5 plays a

283

positive role in ethylene-mediated etiolated hypocotyl elongation, we investigated

284

the effects of WDL5 on regulation of cortical microtubule organization in response

285

to ethylene. Wild-type and wdl5-1 seedlings were grown for 4 days in the dark and

286

treated with ACC. After 4 days, parallel arrays of cortical microtubules were mostly

287

transversely oriented to the longitudinal hypocotyl growth axis in the upper regions

288

of etiolated hypocotyls in wildtype and wdl5-1 mutants (Fig. 5A and 5E). After

289

treatment with 100 μM ACC for 40 min, most of the cells in wild-type hypocotyls

290

had random, oblique, or longitudinal microtubule arrays (Fig. 5B and 5I), while

291

almost 40% of transverse cortical microtubules remained in the hypocotyl cells of

292

wdl5-1 seedlings (Fig. 5F and 5I). Increasing the duration of treatment induced

293

predominantly longitudinal cortical microtubules in the wild-type cells, but not

294

wdl5-1 cells (Fig. 5C, 5G, and 5I), indicating that cortical microtubule reorganization

295

was partially hindered in wdl5-1 cells in response to ACC treatment. Cortical

296

microtubule arrays in wild-type and wdl5-1 cells did not differ after the cells were

297

treated with mock buffer for 90 min (Fig. 5D, 5H, and 5I). This demonstrates that the

298

much longer etiolated hypocotyl phenotype in wdl5-1 cells is correlated with a defect 11 Downloaded from www.plantphysiol.org on July 9, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

299

in microtubule reorganization from transverse to longitudinal in response to

300

ethylene.

301

To gain insight into the mechanism by which WDL5 mediates etiolated hypocotyl

302

cell elongation through regulation of cortical microtubule organization in response to

303

ethylene, wild-type and wdl5-1 epidermal hypocotyl cells pretreated with ACC were

304

treated with the microtubule-disrupting drug oryzalin. Epidermal cells in the top

305

region were used to compare cortical microtubule stability. To quantify the effects of

306

oryzalin on cortical microtubules following treatment with ACC, the number of

307

cortical microtubules in each treatment group was determined. The density of

308

cortical microtubules in wild-type epidermal cells was similar to the density in

309

wdl5-1 cells before treatment (Fig. 6A, 6D, 6G, and 6J). However, the relative

310

microtubule numbers were significantly different after oryzalin treatment (Fig. 6M).

311

In the absence of ACC pretreatment, cortical microtubules were mostly disrupted in

312

wdl5-1 epidermal cells after treatment with 10 μM oryzalin for 3 min, while more

313

microtubules were observed in wild-type cells (Fig. 6B and 6E). Statistical analysis

314

using paired Student’s t-test indicated that this difference was significant (Fig. 6M,

315

see blue asterisks), suggesting that WDL5 functions as a microtubule stabilizer in

316

vivo.

317

In addition, the percentage of remaining cortical microtubules increased in

318

wild-type epidermal cells (from ~52% to ~68%) when pretreated with 100 μM ACC

319

for 90 min and then treated with 10 μM oryzalin for 3 min (Fig. 6B, 6H, and 6M, see

320

red asterisks), but slightly increased in wdl5-1 cells (from ~38% to ~43%) (Fig. 6E,

321

6K, and 6M). Increasing the duration of oryzalin treatment resulted in disappearance

322

of most of the cortical microtubules in mock buffer-pretreated and ACC-pretreated

323

wdl5-1 cells. The percentage of remaining cortical microtubules was ~12% and

324

~18%, respectively (Fig. 6F, 6L, and 6M). However, the percentage of cortical

325

microtubules was significantly increased in ACC-pretreated wild-type cells

326

compared with mock buffer-pretreated wild-type cells (from ~20% to ~56%) (Fig.

327

6C, 6I, and 6M, see green asterisks). Statistical analysis using paired Student’s t-test

328

indicated that the differences were significant (Fig. 6M). These results demonstrate 12 Downloaded from www.plantphysiol.org on July 9, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

329

that WDL5 functions as a microtubule stabilizer in ethylene-inhibited etiolated

330

hypocotyl elongation.

331 332 333

WDL5 Binds to and Stabilizes Microtubules in Vitro Given that WDL5 is required for cortical microtubule stability in

334

ethylene-mediated hypocotyl elongation, the molecular basis for WDL5 regulation

335

of microtubules was investigated in vitro. A GST-WDL5-His fusion protein was

336

purified from Escherichia coli and a co-sedimentation assay was performed to

337

determine whether WDL5 directly binds to microtubules. GST-WDL5-His (4

338

μΜ) was incubated with preformed 5 μΜ paclitaxel-stabilized microtubules at room

339

temperature for 20 min, followed by centrifugation. SDS-PAGE analysis revealed

340

that GST-WDL5-His, but not GST alone, bound to and co-sedimented with the

341

microtubules (Fig. 7A). To investigate the localization pattern of WDL5 in vivo, a

342

construct expressing WDL5 fused with a C-terminal GFP tag under control of the

343

35S promoter was generated and transiently introduced into Arabidopsis pavement

344

cells. Confocal microscopy observations showed that WDL5-GFP exhibited

345

filamentous localization in the cells (Fig. 7B). The filamentous localization was

346

disrupted by treatment with oryzalin (Fig. 7D), but were nearly intact in the presence

347

of LatA, a reagent that depolymerizes actin filaments (Fig. 7C). To confirm this

348

result, WDL5-GFP and MBD-mCherry were transiently co-expressed in Arabidopsis

349

pavement cells. Confocal microscopy showed that the WDL5-GFP green fluorescent

350

signal overlapped with the MBD-mCherry red fluorescent signal, confirming that

351

WDL5 co-localized with microtubules (Fig. 7E to 7G). Colocalization was analyzed

352

by plotting WDL5-GFP and MBD-mCherry signal intensities using ImageJ software

353

(Fig. 7G and 7H). Colocalization of WDL5-mCherry with cortical microtubules was

354

also observed in hypocotyl epidermal cells of Arabidopsis seedlings stably

355

expressing WDL5-mCherry in a GFP-tubulin background (Supplemental Fig. S5).

356

These data demonstrate that WDL5 colocalizes with microtubules in vitro and in

357

cells.

358

To investigate the direct effect of WDL5 on microtubule stability, low temperature 13 Downloaded from www.plantphysiol.org on July 9, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

359

and dilution treatments that disrupt microtubules were applied in vitro. MAP65-1,

360

which stabilizes microtubules under these conditions (Mao et al., 2005), was used as

361

a control. Rhodamine-labeled tubulin (20 μM) was incubated in the presence or

362

absence of WDL5 (3 μM) or MAP65-1 (3 μM) to allow tubulin polymerization (Fig.

363

8A to 8C). The solutions were then incubated at 10°C for 30 min (Fig. 8D to 8F) or

364

diluted with 50× pre-warmed buffer and incubated at 35°C for 60 min (Fig. 8G to 8I)

365

prior to fixation. After fixation, samples were observed by confocal microscopy.

366

Like WVD2 and WDL3 in the Arabidopsis WVD2/WDL protein family, WDL5

367

fusion proteins induced formation of large microtubule bundles in vitro (Fig. 8B and

368

Supplemental Fig. S6). Microtubule filaments in the absence of WDL5 were fully

369

disassembled after low temperature and dilution treatments (Fig. 8D and 8G).

370

However, many large microtubule bundles remained in the presence of WDL5 (Fig.

371

8E and 8H) or MAP65-1 (Fig. 8F and 8I) after the treatments. These results indicate

372

that WDL5 is capable of stabilizing microtubules against low temperature- and

373

dilution-induced depolymerization.

374 375 376 377 378

DISCUSSION Understanding how hormone signaling regulates cortical microtubules is essential

379

in elucidating developmental mechanisms in plants. In this study, we demonstrated

380

that the microtubule-stabilizing protein WDL5 participates in ethylene

381

signaling-inhibited etiolated hypocotyl cell elongation.

382 383

Hormone Signaling Pathway Directly Regulates Microtubule-Associated

384

Proteins in Hypocotyl Elongation

385

Many phytohormones play crucial roles in regulating hypocotyl elongation, such

386

as gibberellins (GAs), auxin, brassinosteroid (BR) and ethylene. Microarray assays

387

have shown that the transcriptional levels of many microtubule-associated proteins

388

are regulated by hormones (Zhong et al., 2009; Sun et al., 2010). However, it is still 14 Downloaded from www.plantphysiol.org on July 9, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

389

unclear whether those proteins are involved in hormone signaling-mediated

390

hypocotyl elongation. A previous study showed that Arabidopsis MICROTUBULE

391

DESTABILIZING PROTEIN40 (MDP40) promotes etiolated hypocotyl cell

392

elongation via BR signaling (Wang et al., 2012). In the present study, we showed that

393

WDL5 participates in ethylene signaling-inhibited etiolated hypocotyl cell

394

elongation. Although other hormones, such as GAs and auxin, are capable of altering

395

cortical microtubule organization in growing cells (Nick et al., 1990; Shibaoka, 1993,

396

1994; Fujino et al., 1995; Vineyard et al., 2013), no microtubule-associated proteins

397

have been identified that target and are regulated by their signaling pathways. Thus,

398

investigating individual hormone signaling pathways through microtubules by

399

directly targeting microtubule-associated proteins in hypocotyl elongation is

400

important in understanding regulatory mechanisms.

401

Previous studies have shown that ethylene and BR signaling pathways play

402

different roles in etiolated hypocotyl cells (Wang et al., 2002; An et al., 2010).

403

Although BRs crosstalk with ethylene in a broad spectrum of physiological and

404

developmental processes (Choudhary et al., 2012), it is still unknown how plant cells

405

coordinate opposite functions on microtubules within the same cell to promote or

406

inhibit elongation. Future studies will be necessary to provide more experimental

407

data to demonstrate whether similar regulatory mechanisms in microtubules are

408

exploited by other environmental and developmental cues and how those pathways

409

crosstalk to mediate plant cell growth and morphogenesis via microtubules.

410 411

Microtubule-Stabilizing Proteins Are Involved in Ethylene-Regulated Etiolated

412

Hypocotyl Cell Elongation

413

Microtubule-associated proteins play positive and negative roles in hypocotyl cell

414

elongation (Li et al., 2011; Liu et al., 2013). These proteins are considered to be

415

microtubule stabilizers or destabilizers depending on their effect on stability (Heald

416

and Nogales, 2002). In this study, cortical microtubule stability increased in etiolated

417

hypocotyl epidermal cells from ACC-treated wild-type seedlings (Fig. 6).

418

Coincidentally, expression of the microtubule stabilizer WDL5 was significantly 15 Downloaded from www.plantphysiol.org on July 9, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

419

increased by treatment with ACC (Fig. 1), and etiolated hypocotyl cell elongation in

420

wdl5-1 was less sensitive to ACC treatment (Fig. 2). Additionally, 16 of the

421

WDL5-overexpressing lines obtained exhibited the shorter-etiolated-hypocotyl

422

phenotype (data not shown), and line10 was selected for analysis. Observation of

423

5-day dark-grown seedlings from the line 10 revealed that the etiolated hypocotyl

424

length was considerably reduced (Supplemental Fig. S7A-C). This evidence suggests

425

that ethylene-inhibited etiolated hypocotyl cell growth may be required to increase

426

levels of negative regulators that function as microtubule stabilizers.

427

Our findings are in agreement with previous studies showing that microtubules are

428

more stable in shorter etiolated hypocotyl cells from some mutants, such as

429

regulatory particle non-ATPase (RPN) subunits RPN10 partial loss-of-function

430

mutant rpn10-1 and a BR-deficient mutant de-etiolated-2 (det2), than in the wildtype

431

(Wang et al., 2009; Wang et al., 2011; Wang et al., 2012). Increasing the expression

432

of microtubule-stabilizers, such as WDL3, also inhibits hypocotyl cell elongation

433

(Liu et al., 2013). Destabilization of cortical microtubules and increased expression

434

of microtubule-destabilizers are necessary for BR promotion of etiolated hypocotyl

435

elongation (Wang et al., 2012). Thus, regulating expression of

436

microtubule-stabilizers and -destabilizers may play a crucial role in

437

hormone-mediated hypocotyl cell elongation. However, the molecular mechanisms

438

involved are complicated. For example, whether the microtubule-stabilizing or

439

-destabilizing activity of those regulators is transient or prolonged in nature and the

440

means through which they coordinate to maintain a dynamic cortical microtubule

441

array in response to diverse hormone signaling are still unclear.

442

In addition, this study showed that decreased WDL5 expression only partially

443

suppressed shorter etiolated hypocotyls in the eto1-1 mutant (Fig. 4). A possible

444

explanation for this phenomenon is that other MAPs with WDL5-like activities are

445

also involved in ethylene-inhibited etiolated hypocotyl elongation. In addition to

446

MAP involvement, microarray assays have shown that expression levels of many

447

negative regulators of plant cell elongation, such as RALF23, RALF31 and RALF33

448

from the rapid alkalinization factor (RALF) family, are obviously upregulated by 16 Downloaded from www.plantphysiol.org on July 9, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

449

ethylene (Srivastava et al., 2009; Zhong et al., 2009; Morato do Canto et al., 2014),

450

which may also be a cause of shorter etiolated hypocotyls in eto1-1. Thus, future

451

studies will be necessary to functionally identify other MAPs and negative regulators

452

of cell elongation involved in ethylene-mediated hypocotyl growth.

453

Ethylene stimulates hypocotyl elongation of Arabidopsis seedlings in the light. A

454

previous study indicated that EIN3 and EIL1 are required for ethylene-promoting

455

hypocotyl elongation in the light, mainly through activation of transcription factor

456

phytochrome-interacting factor 3 (PIF3) (Zhong et al., 2012). We did not find the

457

typical PIF3-binding motif (G box) (Monte et al., 2004) in the WDL5 promoter

458

sequence. Although WDL5 expression was found to be increased in light-grown

459

EIN3 overexpressing (EIN3ox) seedlings and decreased in the ethylene-insensitive

460

mutant ein2-5 compared to the wildtype (Supplemental Fig. S8A), hypocotyl

461

elongation in the wdl5-1 mutant was similar as that of wild-type seedlings in the

462

absence or presence of ACC (Supplemental Fig. S8B to S8D), suggesting that

463

WDL5 may not be involved in ethylene-promoted hypocotyl elongation in the light.

464

The potential physiological function of WDL5 in response to ethylene in the light

465

should be further investigated.

466

Characterization of WDL5 provides strong evidence for the role of microtubules

467

as a link between ethylene signaling and ethylene-mediated etiolated hypocotyl cell

468

elongation. We propose the following model describing the function of WDL5 in

469

ethylene-inhibited hypocotyl cell elongation in the dark (Fig. 8J): ethylene functions

470

through a well-defined signal transduction pathway to activate EIN3/EIL1

471

transcription factors; EIN3 directly targets the WDL5 promoter to upregulate WDL5

472

expression; and WDL5 acts on cortical microtubules with microtubule stabilizing

473

activity to maintain a longitudinal organization, which inhibits etiolated hypocotyl

474

cell elongation.

475 476 477

METHODS

478

Plant Materials and Growth Conditions 17 Downloaded from www.plantphysiol.org on July 9, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

479

All plant materials used in this study were from the Arabidopsis thaliana Columbia

480

(Col) ecotype background. The wdl5-1 (CS436432) and wdl5-2 (CS434701) mutants,

481

ordered from ABRC, were from the Col ecotype background. PCR genotyping and

482

sequencing results revealed that wdl5-1 and wdl5-2 are knockout mutants with a

483

T-DNA insertion in the seventh intron and eighth exon of WDL5. Seeds were

484

sterilized and placed on MS medium (Sigma-Aldrich) with 1% agar and 3% sucrose.

485

For hypocotyl measurement, plates were placed at 22°C in the light for 12 h after

486

stratification at 4°C for 2 d and then transferred to the dark for 5 d. Mutants ein2-5

487

(Alonso et al., 1999), eto1-1 (Kieber et al., 1993), and 35S:Tubulin5A-YFP transgenic

488

plants (Kirik et al., 2012) were used in this study.

489 490

Isolation of WDL5 cDNA Clones from Arabidopsis

491

The full-length WDL5 cDNA sequence was amplified using RT-PCR. The primers

492

used to amplify WDL5 were 5′-TCTAGAATGGACCCTGAGAGTATCATGGC-3′

493

and 5′-GGTACCTTAATGCTCAACAGCAACCGC-3′. GST-WDL5-His-tagged

494

fusion proteins were expressed and purified according to the manufacturer’s

495

protocols. Protein concentration was determined using a Bio-Rad protein assay kit.

496

Protein samples were analyzed by SDS-PAGE.

497 498

PWDL5:WDL5 Construction in Arabidopsis

499

To complement the hypocotyl phenotype of the wdl5-1 mutant, a fragment 2138 bp

500

upstream of the initiation codon (ATG) in WDL5 to the stop codon (TAA) was

501

amplified and reconstructed into a pCAMBIA1300 vector. cDNAs for WDL5 were

502

amplified and reconstructed into the expression vector pCAMBIA1300 under the

503

control of the WDL5 promoter and nopaline synthase terminator. Constructs were

504

transformed into Arabidopsis plants by Agrobacterium (strain GV3101).

505

Homozygous lines were used for subsequent analyses.

506 507

ACC Treatment

508

Four-day-old etiolated hypocotyls from wildtype and wdl5-1 with a 18 Downloaded from www.plantphysiol.org on July 9, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

509

35S:Tubulin5A-YFP background grown on MS medium were used. Seedlings were

510

treated with ACC at a concentration of 100 μM for 0, 40 and 90 min, and cortical

511

microtubules were observed using confocal microscopy.

512 513

Microtubule Co-sedimentation Assay

514

Porcine brain tubulins were purified using a previously published method by

515

Castoldi and Popov (2003) and used for sedimentation assays. Tubulin assembly and

516

co-sedimentation of microtubules with GST-WDL5-His fusion proteins were

517

performed as described by Mao et al. (2005) and Li et al. (2011). Purified proteins

518

were centrifuged at 150,000 g at 4°C for 20 min before use. Prepolymerized,

519

paclitaxel-stabilized microtubules (5 μM) were incubated with 3 μM WDL5 fusion

520

proteins in PEM buffer (1 mM MgCl2, 1 mM EGTA, and 100 mM PIPES-KOH, pH

521

6.9) plus 20 μM paclitaxel at room temperature for 20 min. After centrifugation at

522

100,000 g for 20 min, the supernatant and pellets were subjected to SDS-PAGE.

523 524

Low Temperature and Dilution Assays

525

Purified tubulin was conjugated to 5-(and 6-)carboxytetramethylrhodamine

526

succinimidyl ester (NHS)-rhodamine as previously reported (Hyman, 1991).

527

NHS-rhodamine-labeled tubulin underwent an additional round of assembly/

528

disassembly with 30% (v/v) glycerin prior to storage in liquid nitrogen.

529

GST-WDL5-His protein or GST-MAP65-1 protein (3 μM) were added to 20 μM

530

rhodamine-labeled tubulin in PEM buffer containing 1 mM GTP. After tubulin

531

assembly at 35°C for 40 min, the temperature was immediately decreased to 10°C

532

and maintained for 30 min for low temperature experiments. For dilution treatments,

533

the assembled tubulin samples described were diluted with 50× prewarmed PEM

534

buffer containing WDL5 or MAP65-1 and incubated for 60 min at 35°C prior to

535

fixation. Samples were fixed with 1% glutaraldehyde for observation by confocal

536

microscopy.

537 538

PCR Analysis 19 Downloaded from www.plantphysiol.org on July 9, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

539

RT-PCR and quantitative real-time PCR analysis was performed to assess WDL5

540

transcript levels in wild-type, wdl5-1, wdl5-2, eto1-1, and ein2-5 seedlings. Total RNA

541

was isolated using TRIzol reagent (Invitrogen) from hypocotyls of 5-day-old

542

seedlings grown in the dark. Three independent pairs of primers were used to

543

determine the levels of full-length WDL5 transcripts

544

(5′-ATGGACCCTGAGAGTATCATGGC-3′ and

545

5′-TTAATGCTCAACAGCAACCGC-3′), partial WDL5 transcripts located upstream

546

of the T-DNA insertion site (5′-AAGTCAGAATGAGAATTCGGCAAAC-3′ and

547

5′-CATCGTCTGCTTTCGGACTATTAGA-3′) and partial WDL5 transcripts located

548

downstream of the T-DNA insertion site

549

(5′-CTTTTATCAAGAACCTCAGCCGCCT-3′ and

550

5′-TTAATGCTCAACAGCAACCGCTTCA-3′) in wdl5-1 and wdl5-2 mutants. UBQ

551

was amplified as a loading control using the following primers:

552

5′-GACCATAACCCTTGAGGTTGAATC-3′ and

553

5′-AGAGAGAAAGAGAAGGATCGATC-3′.

554

For quantitative real-time PCR, an ABI 7500 real-time PCR system (Applied

555

Biosystems) was used according to the manufacturer’s instructions. Primers used for

556

subsequent detection of WDL5 expression were 5′-

557

AAATGGTTCTGTTGCTCCTAATGTA-3′ and

558

5′-TTTGAGACTTTGGTTTCACCTTCT-3′. UBQ11 was used as an internal control

559

(5′- GCAGATTTTCGTTAAAACC -3′ and 5′-CCAAAGTTCTGCCGTCC-3′).

560

Three biological replicates and 2 to 3 technical replicates (for each biological

561

replicate) were used for each treatment. The average and standard deviation were

562

calculated from the biological replicates.

563 564

EMSA

565

EMSA was performed according to Zhang et al. (2012). Briefly, the recombinant

566

GST-EIN3 141-352 truncated protein was purified from Escherichia coli according

567

to the manufacturer’s instructions. Biotin-labeled DNA fragments were synthesized

568

and used as probes, and biotin-unlabeled DNA fragments of the same sequences 20 Downloaded from www.plantphysiol.org on July 9, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

569

were used as competitors. Nucleotide sequences of the double-stranded

570

oligonucleotides for WDL5 P1 were 5′-TTTTTTTGCCAACCACTTATGTCT-3′ and

571

5′-GTACATTGCGATTTTCAACCTTAAA-3′; WDL5 P2:

572

5′-GATTTAATTCTTTTGGCCTACC-3′ and

573

5′-ATCAACAATATTTCAAAGTTGGAAT -3′; and WDL5 P3: 5′-

574

ACGAAAAGTTTATACCGTTT -3′ and

575

5′-GTCCAAATTAATACTTGTTATAAAA-3′. Primers were labeled using the Biotin

576

5′ End DNA labeling kit (Pierce). Standard reaction mixtures (20 μL) for EMSA

577

contained 1 μg purified proteins, 2 μL biotin-labeled annealed oligo nucleotides, 2

578

μL 10× binding buffer (100 mM Tris, 500 mM KCl, and 10 mM DTT, pH 7.5), 1 μL

579

50% glycerol, 1 μL 1% Nonidet P-40, 1 μL 1 M KCl, 1 μL 100 mM MgCl2, 1 μL

580

200 mM EDTA, 1 μL 1 mg/mL poly (dI-dC), and 10 μL ultrapure water. Reactions

581

were incubated at room temperature (25°C) for 30 min and loaded onto a 6% native

582

polyacrylamide gel in TBE buffer (45 mM Tris, 45 mM boric acid, and 1 mM EDTA,

583

pH 8.3). The gel was sandwiched and transferred to an N+ nylon membrane

584

(Millipore) in 0.5×TBE buffer at 380 mA at 4°C for 60 min. Detection of

585

biotin-labeled DNA by chemiluminescence was performed based on the instructions

586

provided in the Light Shift Chemiluminescent EMSA kit (Pierce).

587 588

Chromatin Immunoprecipitation (ChIP)

589

Five-day-old dark-grown seedlings were treated with 10 µM β-estradiol or DMSO as

590

a control under the same growth conditions for 4 h. ChIP was performed as

591

previously described (Johnson et al., 2002) using an anti-FLAG monoclonal

592

antibody (Sigma) for immunoprecipitation. Equal quantities of starting plant material

593

and ChIP reagents were used for the PCR reaction. Primers used to detect the EIN3

594

target WDL5 promoter were P1: 5′-TTTTTTTGCCAACCACTTATGTCT-3′ and

595

5′-GTACATTGCGATTTTCAACCTTAAA-3′; P2:

596

5′-GATTTAATTCTTTTGGCCTACC-3′ and

597

5′-ATCAACAATATTTCAAAGTTGGAAT-3′; and actin2 as a control

598

(5′-GGTAACATTGTGCTCAGTGGTGG-3′ and 21 Downloaded from www.plantphysiol.org on July 9, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

599

5′-AACGACCTTAATCTTCATGCTGC-3′). ChIP experiments were performed

600

independently 3 times.

601 602

Ballistics-Mediated Transient Expression in Leaf Epidermal Cells

603

Subcellular localization of WDL5-GFP and cortical microtubules was visualized

604

using transiently expressed 35S:WDL5-GFP and 35S:MBD-mCherry constructs in

605

Arabidopsis (Columbia ecotype) leaf epidermal cells. Experiments were performed

606

as previously described by Fu et al. (2002). One microgram of 35S:WDL5-GFP and

607

1 μg of 35S:MBD-mCherry DNA were used for particle bombardment. Six to 8 h

608

after bombardment, GFP and mCherry signals were detected using a Zeiss LSM 510

609

META confocal microscope (Zeiss, Jena, Germany). Filamentous structures

610

containing WDL5-GFP in leaf epidermal cells were visualized after treatment with

611

10 μM oryzalin and 100 nM LatA for 30 min.

612 613

Quantification of Cortical Microtubules in the Cell

614

ImageJ software (http://rsb.info.nih.gov/ij/) was used to quantify the density of

615

cortical microtubules in the cell. A vertical line that oriented to the majority of the

616

cortical microtubules with a fixed length (~10 μm) was drawn, and the density of

617

cortical microtubules across the line was measured. Four repeated measures were

618

performed for each cell, and at least 36 cells from each treatment were used. The

619

values were recorded and significance was analyzed using the paired Student’s t test.

620 621 622

Accession Numbers

623

Sequence data can be found in the Arabidopsis Genome Initiative under accession

624

numbers WDL5, At4g32330.

625 626 627

ACKNOWLEDGEMENTS

628

The authors thank Dr. Ming Yuan (China Agricultural University) for critical reading 22 Downloaded from www.plantphysiol.org on July 9, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

629

and comments on the article and Dr. Hongwei Guo (Peking University) and Dr.

630

Shuhua Yang (China Agricultural University) for generously providing the

631

ethylene-related Arabidopsis mutant seeds.

632 633 634

AUTHOR CONTRIBUTIONS

635

T.M. designed the project. J.S. and Q.M. performed specific experiments and

636

analyzed the data. T. M. wrote, revised and edited the manuscript.

637

23 Downloaded from www.plantphysiol.org on July 9, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

638

FIGURE LEGENDS

639 640

Figure 1. Ethylene Upregulates WDL5 Expression.

641

(A) WDL5 expression was determined using quantitative real-time PCR with RNA

642

purified from the wildtype, eto1-1 or ein2-5 etiolated seedlings. Wildtype gene

643

expression levels were set to 1. The data represent the mean ± SD of three

644

independent experiments. Significant differences from the corresponding wildtype

645

are indicated by an asterisk (**P < 0.01), as determined by Student’s t-test. (B)

646

Quantitative real-time PCR analysis of WDL5 RNA levels in 4-day-old dark-grown

647

seedlings after various treatment durations using 100 μM ACC or a mock buffer.

648

UBQ11 was used as a reference gene. Gene expression levels in untreated seedlings

649

were set to 1. The data represent the mean ± SD of three independent experiments.

650

Significant differences from corresponding untreated seedlings are indicated by an

651

asterisk (**P < 0.01), as determined by Student’s t-test.

652 653

Figure 2. WDL5 Is a Positive Regulator of Ethylene-inhibited Etiolated Hypocotyl

654

Cell Elongation.

655

(A) Physical structure of Arabidopsis WDL5. WDL5 contains eight exons and seven

656

introns, which are represented by filled boxes and lines, respectively. Positions of

657

two T-DNA insertion mutants, wdl5-1 (T-DNA line CS436432, intron 7) and wdl5-2

658

(T-DNA line CS434701, exon 8), are noted by arrows above the diagram. (B)

659

RT-PCR analysis of WDL5 transcripts in the wildtype Columbia ecotype (Col)

660

seedlings and wdl5-1 mutant, with UBQ as a control. (C) The wdl5-1 mutant shows

661

much longer etiolated hypocotyls when grown on MS for 5 days in the presence of

662

10 μM ACC. (D) Relative hypocotyl length of seedlings grown on MS medium

663

supplemented with 0, 0.25, 0.5, 1, 1.5, 2, 3, 5, 10 and 20 μM ACC in the dark for 5

664

days. Three independent experiments were performed with similar results, each with

665

three biological repeats. More than 40 seedlings were measured in each replicate.

666

t-test, **P < 0.01. Error bars represent the mean ± SE. (E) Scanning electron

667

microscopy images of etiolated hypocotyl epidermal cells from wild-type and wdl5-1

668

seedlings when grown on MS for 5 days in the absence and presence of 10 μM ACC.

669

(F) Relative hypocotyl cell length of wildtype and wdl5-1 was measured and

670

calculated from at least 500 cells under dark growth conditions. t-test, **P < 0.01, 24 Downloaded from www.plantphysiol.org on July 9, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

671

Error bars represent the mean ± SE. Bar in (E) = 100 μm.

672 673

Figure 3. WDL5 Is an EIN3 Target Gene.

674

(A) Three distinctive EIN3-binding sites were predicted in the promoter region of

675

the WDL5 gene. The numbers -389, -1111, and -1205 represented the starting

676

nucleotide position of each EBSs upstream of the transcription start site in the WDL5

677

promoter. Fragment P1 contained one EBS (-389– -393) and located from -332 to

678

-510; fragment P2 contained two close EBSs (-1111– -1115 and -1205– -1209) and

679

located from -1086 to -1247; fragment P3 located from -536 to -690 without putative

680

EBSs upstream of the transcription start site in the WDL5 promoter. (B)

681

ChIP-qRT-PCR assay of EIN3 binding to WDL5 promoters in vivo. Chromatin from

682

dark-grown EIN3-3×FLAG transgenic seedlings was immunoprecipitated with an

683

anti-FLAG antibody, and the amount of the indicated DNA in the immune complex

684

was determined by qRT-PCR. DNA precipitated without addition of the antibody

685

(-Ab) as a negative control. At least three independent experiments were performed

686

with similar results. Data are the mean values of three replicates ± SD from one

687

experiment.

688

EMSA assay for EIN3 binding to WDL5 promoters. Each biotin-labeled DNA

689

fragment was incubated with the GST-EIN3 protein. Competition for labeled

690

promoter sequences was performed by adding an excess of unlabeled probes. A

691

biotin-labeled DNA fragment (P3) that does not contain putative EBSs in the WDL5

692

promoter served as a negative control. The arrow indicates bands resulting from

693

EIN3 binding to P1 (C) and P2 (D) fragments in the WDL5 promoter.

694 695

Figure 4. Decreasing WDL5 Expression Partially Rescues Shorter Etiolated

696

Hypocotyls in the eto1 Mutant.

697

(A) RT-PCR analysis of WDL5 transcripts in wildtype, eto1-1 and wdl5-1eto1-1

698

double mutant seedlings. (B) The wdl5-1eto1-1 double mutant shows longer

699

etiolated hypocotyls than eto1-1 grown on MS in the dark for 5 days. (C) The graph

700

shows the average hypocotyl length measured from at least 40 seedlings under dark

701

growth conditions. (**P < 0.01, t-test). Error bars indicate the mean ± SD. (D)

702

Scanning electron microscopy images of etiolated hypocotyl epidermal cells of

703

wildtype, eto1-1 and wdl5-1eto1-1 double mutants. (E) Length of etiolated

704

hypocotyl cells from wildtype, eto1-1 and wdl5-1eto1-1 double mutants grown in the 25 Downloaded from www.plantphysiol.org on July 9, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

705

dark for 5 days. t-test, **P < 0.01, Error bars represent the mean ± SD. Bar in (D) =

706

100 μm.

707 708

Figure 5. Organization of Cortical Microtubules in wdl5-1 Cells Is Insensitive to

709

Treatment with ACC.

710

Wildtype (A-D) and wdl5-1 mutant (E-H) etiolated hypocotyls with a YFP-tubulin

711

background were treated with mock buffer or 100 μM ACC. Cortical microtubules

712

from the upper region of hypocotyl epidermal cells were observed. (A) and (E),

713

without ACC treatment; (B) and (F), treated with ACC for 40 min; (C) and (G),

714

treated with ACC for 90 min; (D) and (H), treated with a mock buffer for 90 min. (I)

715

Frequency of microtubule orientation patterns in the upper region of etiolated

716

hypocotyl epidermal cells from the wildtype and wdl5-1 mutant (n > 120 cells). Bar

717

in (H) = 20 μm.

718 719

Figure 6. Cortical Microtubules Are More Sensitive to Treatment with Oryzalin in

720

wdl5-1 Cells in Response to ACC.

721

Cortical microtubules were observed in the upper region of etiolated hypocotyl

722

epidermal cells from the wildtype (A-C and G-I) and wdl5-1 mutant (D-F and J-L)

723

with a YFP-tubulin background pretreated with mock buffer or 100 μM ACC after

724

treatment with 0 μM (A, D, G, and J) or 10 μM oryzalin for 3 min (B, E, H, and K)

725

or 8 min (C, F, I, and L). (M) Relative number of cortical microtubules in hypocotyl

726

epidermal cells from the wildtype and wdl5-1 using ImageJ software (n > 50 cells

727

from each sample). T tests compared the number of cortical microtubules in

728

hypocotyl epidermal cells of wdl5-1 with the number of cortical microtubules in the

729

wildtype under the same conditions. **P < 0.01, *P < 0.05, t-test. Error bars

730

represent the mean ± SD. Bar in (L) = 20 μm.

731 732

Figure 7. WDL5 Directly Binds to Microtubules.

733

(A) GST-WDL5-His was co-sedimented with paclitaxel-stabilized microtubules.

734

GST-WDL5-His was most abundant in the supernatant (S) in the absence of

735

microtubules (MTs), but co-sedimented with microtubules into pellets (P).

736

WDL5 colocalizes with cortical microtubules in cells. (B) WDL5-GFP was

737

transiently expressed in Arabidopsis pavement cells, where it localized to 26 Downloaded from www.plantphysiol.org on July 9, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

738

filamentous structures. The filamentous pattern of WDL5-GFP was essentially

739

unaffected when treated with 100 nM LatA for 30 min (C), but was disrupted when

740

the cells were treated with 10 μM oryzalin for 30 min (D). (E) to (G) Analysis of

741

colocalization of transiently expressed WDL5-GFP and MBD-mCherry. (H) Plot of a

742

line scan drawn in (G) showing a strong correlation between spatial localization of

743

WDL5-GFP and MBD-mCherry. Bar in (G) = 20 μm.

744 745

Figure 8. WDL5 Stabilizes Microtubules in Vitro.

746

(A) to (C) Images of microtubules polymerized from rhodamine-labeled tubulin (20

747

μM) incubated in the presence or absence of 3 μM GST-WDL5-His or

748

GST-MAP65-1 protein for 30 min. Samples from (A) to (C) were subjected to 10°C

749

for 30 min ([D] to [F]) or diluted with a solution containing WDL5 or MAP65-1 in

750

50× PEM buffer ([G] to [I]). (J) Model of WDL5 functions on cortical microtubules

751

in ethylene-inhibited etiolated hypocotyl cell elongation. Ethylene activates the

752

transcription factor EIN3/EIL1 by a well-defined signal transduction pathway; EIN3

753

directly regulates WDL5 expression; WDL5 alters the stability of and reorganizes

754

cortical microtubules, which results in inhibition of etiolated hypocotyl cell

755

elongation. Arrows and bars represent positive and negative regulations, respectively.

756

Bar in (I) = 20 μm.

757 758

Supplemental Figure S1. Identification of Partial WDL5 Transcripts in wdl5-1 and

759

wdl5-2 Mutants.

760

Transcript expression levels upstream and downstream of the T-DNA insertion

761

position were detected by RT-PCR using two independent primers in wdl5-1 and

762

wdl5-2 mutants. Partial transcript 1 is located upstream of the T-DNA insertion site

763

for WDL5, and partial transcript 2 is located downstream of the T-DNA insertion site

764

in wdl5-1 and wdl5-2 mutants. UBQ was used as a control. RT-PCR results show that

765

partial transcripts upstream of the insertion position were detected in wdl5-1 and

766

wdl5-2 mutants.

767 768

Supplemental Figure S2. Abnormal Etiolated Hypocotyl Elongation in a Different

769

WDL5 T-DNA Insertion Line in Response to ACC.

770

(A) RT-PCR analysis of WDL5 transcripts in wildtype Columbia ecotype (Col) 27 Downloaded from www.plantphysiol.org on July 9, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

771

seedlings and wdl5-2 (CS434701) Arabidopsis, with UBQ as a control. (B) Wildtype

772

and wdl5-2 mutant seedlings were grown on MS with or without 10 μM ACC in the

773

dark for 5 days. (C) The graph shows the relative hypocotyl length measured from at

774

least 45 seedlings grown on MS medium supplemented with 0 and 10 μM ACC in

775

the dark. Three independent experiments were performed with similar results, each

776

with three biological repeats. t-test, **P < 0.01, error bars represent the mean ± SE, n

777

= 3.

778 779

Supplemental Figure S3. Longer Hypocotyl Phenotype in wdl5-1 Is Completely

780

Suppressed by WDL5 Expression Driven by its Native Promoter in Response to

781

ACC.

782

(A) RT-PCR revealed that WDL5 expression levels were restored in the wdl5-1

783

mutant after transformation with a complementation construct (C-wdl5-1 refers to

784

wdl5-1 transformed with the construct). (B) Wildtype, wdl5-1 mutant and C-wdl5-1

785

seedlings were grown on MS with or without 10 μM ACC in the dark for 5 days. (C)

786

Graphs show the average hypocotyl length measured from at least 120 dark-grown

787

seedlings. C-wdl5-1 had a similar etiolated hypocotyl length as the wildtype,

788

whereas the wdl5-1 mutant had a longer etiolated hypocotyl length when grown on

789

the same medium (t-test, **P < 0.01). Error bars represent the mean ± SD.

790 791 792

Supplemental Figure S4. ERF1 Does not Bind to the WDL5 Promoter.

793

(A) Residues of the WDL5 promoter and mutated WDL5 promoter (mWDL5). Red

794

font represents the ERF1-binding motif with a variant base site, and blue font

795

represents a typical ERF1-binding motif by replacing the base T with C (see asterisk)

796

in the WDL5 promoter sequence. (B) EMSA assay showed that the GST-ERF1

797

protein does not bind to the WDL5 promoter ("Biotin-Probe"), but it does bind to the

798

mutated WDL5 promoter ("Biotin-mProbe", arrow).

799 800 801

Supplemental Figure S5. WDL5-GFP Colocalizes with Cortical Microtubules in

802

Cells.

803

WDL5-mCherry (left) and cortical microtubules (middle) in etiolated hypocotyl cells 28 Downloaded from www.plantphysiol.org on July 9, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

804

from WDL5-mCherry transgenic Arabidopsis seedlings carrying tubulin tagged with

805

GFP. The merged image is shown on the right. Bar = 10 μm.

806 807

Supplemental Figure S6. WDL5 Induces Formation of Microtubule Bundles in

808

Vitro.

809

Fluorescent images are shown in (A) to (D) and electron micrographs are shown in

810

(E) to (F). (A) and (E) Microtubules (MTs) polymerized in the absence of WDL5.

811

(B) and (F) Microtubule bundles induced by GST-WDL5-His.

812

(C) Microtubule bundles induced by WDL5-His without a GST tag.

813

(D) Microtubule bundles induced by GST-WDL5-His were sensitive to NaCl. Single

814

microtubules were observed after treatment with 200 mM NaCl. Bar in (D) = 20 μm.

815

Bar in (F) = 200 nm.

816 817

Supplemental Figure S7. Overexpression of WDL5 Suppresses Etiolated Hypocotyl

818

Elongation.

819

(A) RT-PCR analysis of WDL5 transcript levels in wildtype and WDL5 transgenic

820

seedlings. UBQ was used as a control. (B) WDL5 transgenic seedlings exhibit shorter

821

hypocotyls after growth on MS in the dark for 5 days. The graph shows the average

822

hypocotyl length measured from at least 38 seedlings under dark growth conditions.

823

t-test, **P < 0.01. Error bars represent the mean ± SD.

824 825

Supplemental Figure S8. WDL5 May Not Be Involved in Ethylene-Promoting

826

Hypocotyl Cell Elongation in the Light.

827

(A) WDL5 expression levels were determined using quantitative real-time PCR with

828

RNA purified from light-grown wildtype, ein2-5 and EIN3 overexpression seedlings.

829

Error bars represent ± SD (n = 3). The wdl5-1 mutant shows similar hypocotyl

830

lengths as the wildtype when grown on MS for 7 days in the absence (B) and

831

presence of 10 μM ACC (C). (D) The graph shows the hypocotyl length measured

832

from at least 40 seedlings under light growth conditions. Error bars represent ± SD.

29 Downloaded from www.plantphysiol.org on July 9, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

833 834

REFERENCES Alonso JM, Hirayama T, Roman G, Nourizadeh S, Ecker JR (1999) EIN2, a

835

bifunctional transducer of ethylene and stress responses in Arabidopsis. Science

836

284: 2148–2152

837

An F, Zhao Q, Ji Y, Li W, Jiang Z, Yu X, Zhang C, Han Y, He W, Liu Y, Zhang S,

838

Ecker JR, Guo H (2010) Ethylene-induced stabilization of ETHYLENE

839

INSENSITIVE3 and EIN3-LIKE1 is mediated by proteasomal degradation of

840

EIN3 binding F-box 1 and 2 that requires EIN2 in Arabidopsis. Plant Cell 22:

841

2384–2401

842 843 844 845 846

Bashline L, Lei L, Li S, Gu Y (2014) Cell wall, cytoskeleton, and cell expansion in higher plants. Mol Plant 7: 586–600 Bleecker AB, Estelle MA, Somerville C, Kende H (1988) Insensitivity to ethylene conferred by a dominant mutation in Arabidopsis thaliana. Science 41: 1086–1089 Boutrot F, Segonzac C, Chang KN, Qiao H, Ecker JR, Zipfel C, Rathjen JP (2010)

847

Direct transcriptional control of the Arabidopsis immune receptor FLS2 by the

848

ethylene-dependent transcription factors EIN3 and EIL1. Proc Natl Acad Sci USA

849

107: 14502–14507

850

Castoldia M, Popov A (2003) Purification of brain tubulin through two cycles of

851

polymerization-depolymerization in a high-molarity buffer. Protein Expres Purif

852

32: 83–88

853

Chang KN, Zhong S, Weirauch MT, Hon G, Pelizzola M, Li H, Huang SS,

854

Schmitz RJ, Urich MA, Kuo D, Nery JR, Qiao H, Yang A, Jamali A, Chen H,

855

Ideker T, Ren B, Bar-Joseph Z, Hughes TR, Ecker JR (2013) Temporal

856

transcriptional response to ethylene gas drives growth hormone cross-regulation in

857

Arabidopsis. Elife 00675

858

Chen H, Xue L, Chintamanani S, Germain H, Lin H, Cui H, Cai R, Zuo J, Tang

859

X, Li X, Guo H, Zhou JM (2009) ETHYLENE INSENSITIVE3 and ETHYLENE

860

INSENSITIVE3-LIKE1 repress SALICYLIC ACID INDUCTION DEFICIENT2

861

expression to negatively regulate plant innate immunity in Arabidopsis. Plant Cell

862

21: 2527–2540

863 864 865

Choudhary SP, Yu JQ, Yamaguchi-Shinozaki K, Shinozaki K, Tran LS (2012) Benefits of brassinosteroid crosstalk. Trends Plant Sci 17: 594–605 Crowell EF, Timpano H, Desprez T, Franssen-Verheijen T, Emons AM, Höfte H, 30 Downloaded from www.plantphysiol.org on July 9, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

866

Vernhettes S (2011) Differential regulation of cellulose orientation at the inner and

867

outer face of epidermal cells in the Arabidopsis hypocotyls. Plant Cell 23:

868

2592–2605

869 870 871

Ecker JR (1995) The ethylene signal transduction pathway in plants. Science 268: 667–675 Fan X, Sun Y, Cao D, Bai M, Luo X, Yang H, Wei C, Zhu S, Sun Y, Chong K,

872

Wang Z (2012) BZS1, a B-box protein, promotes photomorphogenesis

873

downstream of both brassinosteroid and light signaling pathways. Mol Plant 5:

874

65–74

875

Fu Y, Li H, Yang Z (2002) The ROP2 GTPase controls the formation of cortical fine

876

F-actin and the early phase of directional cell expansion during Arabidopsis

877

organogenesis. Plant Cell 14: 777–794

878

Fujimoto SY, Ohta M, Usui A, Shinshi H, Ohme-Takagi M (2000) Arabidopsis

879

ethylene-responsive element binding factors act as transcriptional activators or

880

repressors of GCC box-mediated gene expression. Plant Cell 12: 393–404

881

Fujino K, Koda Y, Kikuta Y (1995) Reorientation of cortical microtubules in the

882

sub-apical region during tuberization in single-node stem segments of potato in

883

culture. Plant Cell Physiol 36: 891–895

884

Galva C, Kirik V, Lindeboom JJ, Kaloriti D, Rancour DM, Hussey PJ, Bednarek

885

SY, Ehrhardt DW, Sedbrook JC (2014) The microtubule plus-end tracking

886

proteins SPR1 and EB1b interact to maintain polar cell elongation and directional

887

organ growth in Arabidopsis. Plant Cell 26: 4409–4425

888

Guo H, Ecker JR (2003) Plant responses to ethylene gas are mediated by

889

SCF(EBF1/EBF2)-dependent proteolysis of EIN3 transcription factor. Cell 115:

890

667–677

891

Heald R, Nogales E (2002) Microtubule dynamics. J Cell Sci 115: 3–4

892

Hyman A (1991) Preparation of marked microtubules for the assay of the polarity of

893

microtubule-based motors by fluorescence. J Cell Sci 14 Suppl: 125–127

894

Johnson L, Cao X, Jacobsen S (2002) Interplay between two epigenetic marks.

895

DNA methylation and histone H3 lysine 9 methylation. Curr Biol 12: 1360–1367

896

Kieber JJ, Rothenberg M, Roman G, Feldmann KA, Ecker JR (1993) CTR1, a

897

negative regulator of the ethylene response pathway in Arabidopsis, encodes a

898

member of the raf family of protein kinases. Cell 72: 427–441 31 Downloaded from www.plantphysiol.org on July 9, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

899

Kirik A, Ehrhardt D, Kirik V (2012) TONNEAU2/FASS regulates the geometry of

900

microtubule nucleation and cortical array organization in interphase Arabidopsis

901

cells. Plant Cell 24: 1158–1170

902 903 904

Kosugi S, Ohashi Y (2000) Cloning and DNA-binding properties of a tobacco Ethylene-Insensitive3 (EIN3) homolog. Nucleic Acids Res 28: 960–967 Le J, Vandenbussche F, De Cnodder T, Van Der Straeten D, Verbelen JP (2005)

905

Cell elongation and microtubule behaviour in the Arabidopsis hypocotyl: responses

906

to ethylene and auxin. J Plant Growth Regul 24: 166–178

907

Lei L, Zhang T, Strasser R, Lee CM, Gonneau M, Mach L, Vernhettes S, Kim SH,

908

J Cosgrove D, Li S, Gu Y (2014) The jiaoyao1 mutant is an allele of korrigan1

909

that abolishes endoglucanase activity and affects the organization of both cellulose

910

microfibrils and microtubules in Arabidopsis. Plant Cell 26: 2601–2616

911

Li J, Wang X, Qin T, Zhang Y, Liu X, Sun J, Zhou Y, Zhu L, Zhang Z, Yuan M,

912

Mao T (2011) MDP25, a novel calcium regulatory protein, mediates hypocotyl cell

913

elongation by destabilizing cortical microtubules in Arabidopsis. Plant Cell 23:

914

4411–4427

915

Liu X, Qin T, Ma Q, Sun J, Liu Z, Yuan M, Mao T (2013) Light-regulated

916

hypocotyl elongation involves proteasome-dependent degradation of the

917

microtubule regulatory protein WDL3 in Arabidopsis. Plant Cell 25: 1740–1755

918 919 920 921

Lloyd C (2011) Dynamic microtubules and the texture of plant cell walls. Int Rev Cell Mol Biol 287: 287–329 Lloyd C, Chan J (2008) The parallel lives of microtubules and cellulose microfibrils. Curr Opin Plant Biol 11: 641–646

922

Luo XM, Lin WH, Zhu S, Zhu JY, Sun Y, Fan XY, Cheng M, Hao Y, Oh E, Tian

923

M, Liu L, Zhang M, Xie Q, Chong K, Wang ZY (2010) Integration of light- and

924

brassinosteroid-signaling pathways by a GATA transcription factor in Arabidopsis.

925

Dev Cell 19: 872–883

926

Mao T, Jin L, Li H, Liu B, Yuan M (2005) Two microtubule-associated proteins of

927

the Arabidopsis MAP65 family function differently on microtubules. Plant

928

Physiol 138: 654–662

929 930

Monte E, Tepperman JM, Al-Sady B, Kaczorowski KA, Alonso JM, Ecker JR, Li X, Zhang Y, Quail PH (2004) The phytochrome-interacting transcription factor, 32 Downloaded from www.plantphysiol.org on July 9, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

931

PIF3, acts early, selectively, and positively in light-induced chloroplast

932

development. Proc Natl Acad Sci USA 101: 16091–16098

933

Morato do Canto A, Ceciliato PH, Ribeiro B, Ortiz Morea FA, Franco Garcia AA,

934

Silva-Filho MC, Moura DS (2014) Biological activity of nine recombinant

935

AtRALF peptides: implications for their perception and function in Arabidopsis.

936

Plant Physiol Biochem 75: 45–54

937

Nakajima K, Furutani I, Tachimoto H, Matsubara H, Hashimoto T (2004)

938

SPIRAL1 encodes a plant-specific microtubule-localized protein required for

939

directional control of rapidly expanding Arabidopsis cells. Plant Cell 16:

940

1178–1190

941

Nick P, Bergfeld R, Schafer E, Schopfer P (1990) Unilateral reorientation of

942

microtubules at the outer epidermal wall during photo- and gravitropic curvature of

943

maize coleoptiles and sunflower hypocotyls. Planta 181: 162–168

944

Niwa Y, Yamashino T, Mizuno T (2009) The circadian clock regulates the

945

photoperiodic response of hypocotyl elongation through a coincidence mechanism

946

in Arabidopsis thaliana. Plant Cell Physiol 50: 838–854

947

Perrin RM, Wang Y, Yuen CY, Will J, Masson PH (2007) WVD2 is a novel

948

microtubule-associated protein in Arabidopsis thaliana. Plant J 49: 961–971

949

Polko JK, van Zanten M, van Rooij JA, Marée AF, Voesenek LA, Peeters AJ,

950

Pierik R (2012) Ethylene-induced differential petiole growth in Arabidopsis

951

thaliana involves local microtubule reorientation and cell expansion. New Phytol

952

193: 339–348

953

Potuschak T, Lechner E, Parmentier Y, Yanagisawa S, Grava S, Koncz C,

954

Genschik P (2003) EIN3-dependent regulation of plant ethylene hormone signaling

955

by two arabidopsis F box proteins: EBF1 and EBF2. Cell 115: 679–689

956

Shibaoka H (1993) Regulation by gibberellins of the orientation of cortical

957

microtubules in plant cells. Aust J Plant Physiol 20: 461–470

958

Shibaoka H (1994) Plant hormone-induced changes in the orientation of cortical

959

microtubules—alterations in the cross-linking between microtubules and the

960

plasma-membrane. Annu Rev Plant Physiol Plant Mol Biol 45: 527–544

961

Shi Y, Tian S, Hou L, Huang X, Zhang X, Guo H, Yang S (2012) Ethylene 33 Downloaded from www.plantphysiol.org on July 9, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

962

signaling negatively regulates freezing tolerance by repressing expression of CBF

963

and type-A ARR genes in Arabidopsis. Plant Cell 24: 2578–2595

964

Soga K, Yamaguchi A, Kotake T, Wakabayashi K, Hoson T (2010)

965

1-aminocyclopropane-1-carboxylic acid (ACC)-induced reorientation of cortical

966

microtubules is accompanied by a transient increase in the transcript levels of

967

gamma-tubulin complex and katanin genes in azuki bean epicotyls. J Plant Physiol

968

167: 1165–1171

969 970

Srivastava R, Liu JX, Guo H, Yin Y, Howell SH (2009) Regulation and processing of a plant peptide hormone, AtRALF23, in Arabidopsis. Plant J 59: 930–939

971

Sun Y, Fan X, Cao D, Tang W, He K, Zhu J, He, J, Bai M, Zhu S, Oh E, Patil S,

972

Kim T, Ji H, Wong W, Rhee S, Wang Z (2010) Integration of Brassinosteroid

973

signal transduction with the transcription network for plant growth regulation in

974

Arabidopsis. Dev Cell 19: 765–777

975

Takahashi H, Kawahara A, Inoue Y (2003) Ethylene promotes the induction by

976

auxin of the cortical microtubule randomization required for low-pH-induced root

977

hair initiation in lettuce (Lactuca sativa L.) seedlings. Plant Cell Physiol 44:

978

932–940

979

Vineyard L, Elliott A, Dhingra S, Lucas JR, Shaw SL (2013) Progressive

980

transverse microtubule array organization in hormone-induced Arabidopsis

981

hypocotyl cells. Plant Cell 25: 662–676

982

Wang S, Kurepa J, Smalle JA (2009) The Arabidopsis 26S proteasome subunit

983

RPN1a is required for optimal plant growth and stress responses. Plant Cell

984

Physiol 50: 1721–1725

985

Wang S, Kurepa J, Hashimoto T, Smalle JA (2011) Salt stress-induced disassembly

986

of Arabidopsis cortical microtubule arrays involves 26S proteasome–dependent

987

degradation of SPIRAL1. Plant Cell 23: 3412–3427

988

Wang X, Zhang J, Yuan M, Ehrhardt DW, Wang Z, Mao T (2012) Arabidopsis

989

MICROTUBULE DESTABILIZING PROTEIN40 is involved in brassinosteroid

990

regulation of hypocotyl elongation. Plant Cell 24: 4012–4025

991

Wang Z, Nakano T, Gendron J, He J, Chen M, Vafeados D, Yang Y, Fujioka S,

992

Yoshida S, Asami T, Chory J (2002) Nuclear-localized BZR1 mediates

993

Brassinosteroid-induced growth and feedback suppression of Brassinosteroid

994

biosynthesis. Dev Cell 2: 505–513 34 Downloaded from www.plantphysiol.org on July 9, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

995

Yuen C, Pearlman R, Silo-Suh L, Hilson P, Carroll K, Masson P (2003) WVD2

996

and WDL1 modulate helical organ growth and anisotropic cell expansion in

997

Arabidopsis. Plant Physiol 131: 493–506

998

Zhang C, Xu Y, Guo S, Zhu J, Huan Q, Liu H, Wang L, Luo G, Wang X, Chong

999

K (2012) Dynamics of brassinosteroid response modulated by negative regulator

1000 1001

LIC in rice. PLoS Genet 8: e1002686 Zhang L, Li Z, Quan R, Li G, Wang R, Huang R (2011) An AP2

1002

domain-containing gene, ESE1, targeted by the ethylene signaling component

1003

EIN3 is important for the salt response in Arabidopsis. Plant Physiol 157:

1004

854–865

1005

Zhong S, Shi H, Xue C, Wang L, Xi Y, Li J, Quail PH, Deng XW, Guo H (2012) A

1006

molecular framework of light-controlled phytohormone action in Arabidopsis.

1007

Curr Biol 22: 1530–1535

1008

Zhong S, Shi H, Xue C, We, N, Guo H, Deng XW (2014) Ethylene-orchestrated

1009

circuitry coordinates a seedling's response to soil cover and etiolated growth. Proc

1010

Natl Acad Sci USA 111: 3913–3920

1011

Zhong S, Zhao M, Shi T, Shi H, An F, Zhao Q, Guo H (2009) EIN3/EIL1 cooperate

1012

with PIF1 to prevent photo-oxidation and to promote greening of Arabidopsis

1013

seedlings. Proc Natl Acad Sci USA 106: 21431–2143

35 Downloaded from www.plantphysiol.org on July 9, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

Downloaded from www.plantphysiol.org on July 9, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

Downloaded from www.plantphysiol.org on July 9, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

Downloaded from www.plantphysiol.org on July 9, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

Downloaded from www.plantphysiol.org on July 9, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

Downloaded from www.plantphysiol.org on July 9, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

Downloaded from www.plantphysiol.org on July 9, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

Downloaded from www.plantphysiol.org on July 9, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

Downloaded from www.plantphysiol.org on July 9, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

Parsed Citations Alonso JM, Hirayama T, Roman G, Nourizadeh S, Ecker JR (1999) EIN2, a bifunctional transducer of ethylene and stress responses in Arabidopsis. Science 284: 2148-2152 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

An F, Zhao Q, Ji Y, Li W, Jiang Z, Yu X, Zhang C, Han Y, He W, Liu Y, Zhang S, Ecker JR, Guo H (2010) Ethylene-induced stabilization of ETHYLENE INSENSITIVE3 and EIN3-LIKE1 is mediated by proteasomal degradation of EIN3 binding F-box 1 and 2 that requires EIN2 in Arabidopsis. Plant Cell 22: 2384-2401 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Bashline L, Lei L, Li S, Gu Y (2014) Cell wall, cytoskeleton, and cell expansion in higher plants. Mol Plant 7: 586-600 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Bleecker AB, Estelle MA, Somerville C, Kende H (1988) Insensitivity to ethylene conferred by a dominant mutation in Arabidopsis thaliana. Science 41: 1086-1089 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Boutrot F, Segonzac C, Chang KN, Qiao H, Ecker JR, Zipfel C, Rathjen JP (2010) Direct transcriptional control of the Arabidopsis immune receptor FLS2 by the ethylene-dependent transcription factors EIN3 and EIL1. Proc Natl Acad Sci USA 107: 14502-14507 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Castoldia M, Popov A (2003) Purification of brain tubulin through two cycles of polymerization-depolymerization in a high-molarity buffer. Protein Expres Purif 32: 83-88 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Chang KN, Zhong S, Weirauch MT, Hon G, Pelizzola M, Li H, Huang SS, Schmitz RJ, Urich MA, Kuo D, Nery JR, Qiao H, Yang A, Jamali A, Chen H, Ideker T, Ren B, Bar-Joseph Z, Hughes TR, Ecker JR (2013) Temporal transcriptional response to ethylene gas drives growth hormone cross-regulation in Arabidopsis. Elife 00675 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Chen H, Xue L, Chintamanani S, Germain H, Lin H, Cui H, Cai R, Zuo J, Tang X, Li X, Guo H, Zhou JM (2009) ETHYLENE INSENSITIVE3 and ETHYLENE INSENSITIVE3-LIKE1 repress SALICYLIC ACID INDUCTION DEFICIENT2 expression to negatively regulate plant innate immunity in Arabidopsis. Plant Cell 21: 2527-2540 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Choudhary SP, Yu JQ, Yamaguchi-Shinozaki K, Shinozaki K, Tran LS (2012) Benefits of brassinosteroid crosstalk. Trends Plant Sci 17: 594-605 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Crowell EF, Timpano H, Desprez T, Franssen-Verheijen T, Emons AM, Höfte H, Vernhettes S (2011) Differential regulation of cellulose orientation at the inner and outer face of epidermal cells in the Arabidopsis hypocotyls. Plant Cell 23: 2592-2605 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Ecker JR (1995) The ethylene signal transduction pathway in plants. Science 268: 667-675 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Fan X, Sun Y, Cao D, Bai M, Luo X, Yang H, Wei C, Zhu S, Sun Y, Chong K, Wang Z (2012) BZS1, a B-box protein, promotes photomorphogenesis downstream of both brassinosteroid and light signaling pathways. Mol Plant 5: 65-74 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Fu Y, Li H, Yang Z (2002) The ROP2 GTPase controls the formation of cortical fine F-actin and the early phase of directional cell expansion during Arabidopsis organogenesis. Plant Cell 14: 777-794 Pubmed: Author and Title CrossRef: Author and Title

Downloaded from www.plantphysiol.org on July 9, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

Google Scholar: Author Only Title Only Author and Title

Fujimoto SY, Ohta M, Usui A, Shinshi H, Ohme-Takagi M (2000) Arabidopsis ethylene-responsive element binding factors act as transcriptional activators or repressors of GCC box-mediated gene expression. Plant Cell 12: 393-404 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Fujino K, Koda Y, Kikuta Y (1995) Reorientation of cortical microtubules in the sub-apical region during tuberization in single-node stem segments of potato in culture. Plant Cell Physiol 36: 891-895 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Galva C, Kirik V, Lindeboom JJ, Kaloriti D, Rancour DM, Hussey PJ, Bednarek SY, Ehrhardt DW, Sedbrook JC (2014) The microtubule plus-end tracking proteins SPR1 and EB1b interact to maintain polar cell elongation and directional organ growth in Arabidopsis. Plant Cell 26: 4409-4425 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Guo H, Ecker JR (2003) Plant responses to ethylene gas are mediated by SCF(EBF1/EBF2)-dependent proteolysis of EIN3 transcription factor. Cell 115: 667-677 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Heald R, Nogales E (2002) Microtubule dynamics. J Cell Sci 115: 3-4 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Hyman A (1991) Preparation of marked microtubules for the assay of the polarity of microtubule-based motors by fluorescence. J Cell Sci 14 Suppl: 125-127 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Johnson L, Cao X, Jacobsen S (2002) Interplay between two epigenetic marks. DNA methylation and histone H3 lysine 9 methylation. Curr Biol 12: 1360-1367 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Kieber JJ, Rothenberg M, Roman G, Feldmann KA, Ecker JR (1993) CTR1, a negative regulator of the ethylene response pathway in Arabidopsis, encodes a member of the raf family of protein kinases. Cell 72: 427-441 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Kirik A, Ehrhardt D, Kirik V (2012) TONNEAU2/FASS regulates the geometry of microtubule nucleation and cortical array organization in interphase Arabidopsis cells. Plant Cell 24: 1158-1170 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Kosugi S, Ohashi Y (2000) Cloning and DNA-binding properties of a tobacco Ethylene-Insensitive3 (EIN3) homolog. Nucleic Acids Res 28: 960-967 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Le J, Vandenbussche F, De Cnodder T, Van Der Straeten D, Verbelen JP (2005) Cell elongation and microtubule behaviour in the Arabidopsis hypocotyl: responses to ethylene and auxin. J Plant Growth Regul 24: 166-178 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Lei L, Zhang T, Strasser R, Lee CM, Gonneau M, Mach L, Vernhettes S, Kim SH, J Cosgrove D, Li S, Gu Y (2014) The jiaoyao1 mutant is an allele of korrigan1 that abolishes endoglucanase activity and affects the organization of both cellulose microfibrils and microtubules in Arabidopsis. Plant Cell 26: 2601-2616 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Li J, Wang X, Qin T, Zhang Y, Liu X, Sun J, Zhou Y, Zhu L, Zhang Z, Yuan M, Mao T (2011) MDP25, a novel calcium regulatory protein, mediates hypocotyl cell elongation by destabilizing cortical microtubules in Arabidopsis. Plant Cell 23: 4411-4427 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Downloaded from www.plantphysiol.org on July 9, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

Liu X, Qin T, Ma Q, Sun J, Liu Z, Yuan M, Mao T (2013) Light-regulated hypocotyl elongation involves proteasome-dependent degradation of the microtubule regulatory protein WDL3 in Arabidopsis. Plant Cell 25: 1740-1755 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Lloyd C (2011) Dynamic microtubules and the texture of plant cell walls. Int Rev Cell Mol Biol 287: 287-329 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Lloyd C, Chan J (2008) The parallel lives of microtubules and cellulose microfibrils. Curr Opin Plant Biol 11: 641-646 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Luo XM, Lin WH, Zhu S, Zhu JY, Sun Y, Fan XY, Cheng M, Hao Y, Oh E, Tian M, Liu L, Zhang M, Xie Q, Chong K, Wang ZY (2010) Integration of light- and brassinosteroid-signaling pathways by a GATA transcription factor in Arabidopsis. Dev Cell 19: 872-883 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Mao T, Jin L, Li H, Liu B, Yuan M (2005) Two microtubule-associated proteins of the Arabidopsis MAP65 family function differently on microtubules. Plant Physiol 138: 654-662 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Monte E, Tepperman JM, Al-Sady B, Kaczorowski KA, Alonso JM, Ecker JR, Li X, Zhang Y, Quail PH (2004) The phytochromeinteracting transcription factor, PIF3, acts early, selectively, and positively in light-induced chloroplast development. Proc Natl Acad Sci USA 101: 16091-16098 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Morato do Canto A, Ceciliato PH, Ribeiro B, Ortiz Morea FA, Franco Garcia AA, Silva-Filho MC, Moura DS (2014) Biological activity of nine recombinant AtRALF peptides: implications for their perception and function in Arabidopsis. Plant Physiol Biochem 75: 4554 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Nakajima K, Furutani I, Tachimoto H, Matsubara H, Hashimoto T (2004) SPIRAL1 encodes a plant-specific microtubule-localized protein required for directional control of rapidly expanding Arabidopsis cells. Plant Cell 16: 1178-1190 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Nick P, Bergfeld R, Schafer E, Schopfer P (1990) Unilateral reorientation of microtubules at the outer epidermal wall during photoand gravitropic curvature of maize coleoptiles and sunflower hypocotyls. Planta 181: 162-168 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Niwa Y, Yamashino T, Mizuno T (2009) The circadian clock regulates the photoperiodic response of hypocotyl elongation through a coincidence mechanism in Arabidopsis thaliana. Plant Cell Physiol 50: 838-854 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Perrin RM, Wang Y, Yuen CY, Will J, Masson PH (2007) WVD2 is a novel microtubule-associated protein in Arabidopsis thaliana. Plant J 49: 961-971 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Polko JK, van Zanten M, van Rooij JA, Marée AF, Voesenek LA, Peeters AJ, Pierik R (2012) Ethylene-induced differential petiole growth in Arabidopsis thaliana involves local microtubule reorientation and cell expansion. New Phytol 193: 339-348 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Potuschak T, Lechner E, Parmentier Y, Yanagisawa S, Grava S, Koncz C, Genschik P (2003) EIN3-dependent regulation of plant ethylene hormone signaling by two arabidopsis F box proteins: EBF1 and EBF2. Cell 115: 679-689 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Shibaoka H (1993) Regulation by gibberellins of the orientation of cortical microtubules in plant cells. Aust J Plant Physiol 20: 461470 Downloaded from www.plantphysiol.org on July 9, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Shibaoka H (1994) Plant hormone-induced changes in the orientation of cortical microtubules—alterations in the cross-linking between microtubules and the plasma-membrane. Annu Rev Plant Physiol Plant Mol Biol 45: 527-544 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Shi Y, Tian S, Hou L, Huang X, Zhang X, Guo H, Yang S (2012) Ethylene signaling negatively regulates freezing tolerance by repressing expression of CBF and type-A ARR genes in Arabidopsis. Plant Cell 24: 2578-2595 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Soga K, Yamaguchi A, Kotake T, Wakabayashi K, Hoson T (2010) 1-aminocyclopropane-1-carboxylic acid (ACC)-induced reorientation of cortical microtubules is accompanied by a transient increase in the transcript levels of gamma-tubulin complex and katanin genes in azuki bean epicotyls. J Plant Physiol 167: 1165-1171 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Srivastava R, Liu JX, Guo H, Yin Y, Howell SH (2009) Regulation and processing of a plant peptide hormone, AtRALF23, in Arabidopsis. Plant J 59: 930-939 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Sun Y, Fan X, Cao D, Tang W, He K, Zhu J, He, J, Bai M, Zhu S, Oh E, Patil S, Kim T, Ji H, Wong W, Rhee S, Wang Z (2010) Integration of Brassinosteroid signal transduction with the transcription network for plant growth regulation in Arabidopsis. Dev Cell 19: 765-777 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Takahashi H, Kawahara A, Inoue Y (2003) Ethylene promotes the induction by auxin of the cortical microtubule randomization required for low-pH-induced root hair initiation in lettuce (Lactuca sativa L.) seedlings. Plant Cell Physiol 44: 932-940 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Vineyard L, Elliott A, Dhingra S, Lucas JR, Shaw SL (2013) Progressive transverse microtubule array organization in hormoneinduced Arabidopsis hypocotyl cells. Plant Cell 25: 662-676 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Wang S, Kurepa J, Smalle JA (2009) The Arabidopsis 26S proteasome subunit RPN1a is required for optimal plant growth and stress responses. Plant Cell Physiol 50: 1721-1725 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Wang S, Kurepa J, Hashimoto T, Smalle JA (2011) Salt stress-induced disassembly of Arabidopsis cortical microtubule arrays involves 26S proteasome-dependent degradation of SPIRAL1. Plant Cell 23: 3412-3427 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Wang X, Zhang J, Yuan M, Ehrhardt DW, Wang Z, Mao T (2012) Arabidopsis MICROTUBULE DESTABILIZING PROTEIN40 is involved in brassinosteroid regulation of hypocotyl elongation. Plant Cell 24: 4012-4025 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Wang Z, Nakano T, Gendron J, He J, Chen M, Vafeados D, Yang Y, Fujioka S, Yoshida S, Asami T, Chory J (2002) Nuclear-localized BZR1 mediates Brassinosteroid-induced growth and feedback suppression of Brassinosteroid biosynthesis. Dev Cell 2: 505-513 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Yuen C, Pearlman R, Silo-Suh L, Hilson P, Carroll K, Masson P (2003) WVD2 and WDL1 modulate helical organ growth and anisotropic cell expansion in Arabidopsis. Plant Physiol 131: 493-506 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Zhang C, Xu Y, Guo S, Zhu J, Huan Q, Liu H, Wang L, Luo G, Wang X, Chong K (2012) Dynamics of brassinosteroid response modulated by negative regulator LIC in rice. PLoS Genet 8: e1002686 Downloaded from www.plantphysiol.org on July 9, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Zhang L, Li Z, Quan R, Li G, Wang R, Huang R (2011) An AP2 domain-containing gene, ESE1, targeted by the ethylene signaling component EIN3 is important for the salt response in Arabidopsis. Plant Physiol 157: 854-865 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Zhong S, Shi H, Xue C, Wang L, Xi Y, Li J, Quail PH, Deng XW, Guo H (2012) A molecular framework of light-controlled phytohormone action in Arabidopsis. Curr Biol 22: 1530-1535 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Zhong S, Shi H, Xue C, We, N, Guo H, Deng XW (2014) Ethylene-orchestrated circuitry coordinates a seedling's response to soil cover and etiolated growth. Proc Natl Acad Sci USA 111: 3913-3920 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Zhong S, Zhao M, Shi T, Shi H, An F, Zhao Q, Guo H (2009) EIN3/EIL1 cooperate with PIF1 to prevent photo-oxidation and to promote greening of Arabidopsis seedlings. Proc Natl Acad Sci USA 106: 21431-2143 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title

Downloaded from www.plantphysiol.org on July 9, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.

Supplemental Figure S1. Identification of Partial WDL5 Transcripts in wdl5-1 and wdl5-2 Mutants. Transcript expression levels upstream and downstream of the T-DNA insertion position were detected by RT-PCR using two independent primers in wdl5-1 and wdl5-2 mutants. Partial transcript 1 is located upstream of the T-DNA insertion site for WDL5, and partial transcript 2 is located downstream of the T-DNA insertion site in wdl5-1 and wdl5-2 mutants. UBQ was used as a control. RT-PCR results show that partial transcripts upstream of the insertion position were detected in wdl5-1 and wdl5-2 mutants. Supplemental Figure S2. Abnormal Etiolated Hypocotyl Elongation in a Different WDL5 T-DNA Insertion Line in Response to ACC. (A) RT-PCR analysis of WDL5 transcripts in wildtype Columbia ecotype (Col) seedlings and wdl5-2 (CS434701) Arabidopsis, with UBQ as a control. (B) Wildtype and wdl5-2 mutant seedlings were grown on MS with or without 10 M ACC in the dark for 5 days. (C) The graph shows the relative hypocotyl length measured from at least 45 seedlings grown on MS medium supplemented with 0 and 10 M ACC in the dark. Three independent experiments were performed with similar results, each with three biological repeats. t-test, **P < 0.01, error bars represent the mean ± SE, n = 3. Supplemental Figure S3. Longer Hypocotyl Phenotype in wdl5-1 Is Completely Suppressed by WDL5 Expression Driven by its Native Promoter in Response to ACC. (A) RT-PCR revealed that WDL5 expression levels were restored in the wdl5-1 mutant after transformation with a complementation construct (C-wdl5-1 refers to wdl5-1 transformed with the construct). (B) Wildtype, wdl5-1 mutant and C-wdl5-1 seedlings were grown on MS with or without 10 M ACC in the dark for 5 days. (C) Graphs show the average hypocotyl length measured from at least 120 dark-grown seedlings. C-wdl5-1 had a similar etiolated hypocotyl length as the wildtype, whereas the wdl5-1 mutant had a longer etiolated hypocotyl length when grown on the same medium (ttest, **P < 0.01). Error bars represent the mean ± SD.

Supplemental Figure S4. ERF1 Does not Bind to the WDL5 Promoter. 1

(A) Residues of the WDL5 promoter and mutated WDL5 promoter (mWDL5). Red font represents the ERF1-binding motif with a variant base site, and blue font represents a typical ERF1-binding motif by replacing the base T with C (see asterisk) in the WDL5 promoter sequence. (B) EMSA assay showed that the GST-ERF1 protein does not bind to the WDL5 promoter ("Biotin-Probe"), but it does bind to the mutated WDL5 promoter ("Biotin-mProbe", arrow).

Supplemental Figure S5. WDL5-GFP Colocalizes with Cortical Microtubules in Cells. WDL5-mCherry (left) and cortical microtubules (middle) in etiolated hypocotyl cells from WDL5-mCherry transgenic Arabidopsis seedlings carrying tubulin tagged with GFP. The merged image is shown on the right. Bar = 10 μm. Supplemental Figure S6. WDL5 Induces Formation of Microtubule Bundles in Vitro. Fluorescent images are shown in (A) to (D) and electron micrographs are shown in (E) to (F). (A) and (E) Microtubules (MTs) polymerized in the absence of WDL5. (B) and (F) Microtubule bundles induced by GST-WDL5-His. (C) Microtubule bundles induced by WDL5-His without a GST tag. (D) Microtubule bundles induced by GST-WDL5-His were sensitive to NaCl. Single microtubules were observed after treatment with 200 mM NaCl. Bar in (D) = 20 μm. Bar in (F) = 200 nm. Supplemental Figure S7. Overexpression of WDL5 Suppresses Etiolated Hypocotyl Elongation. (A) RT-PCR analysis of WDL5 transcript levels in wildtype and WDL5 transgenic seedlings. UBQ was used as a control. (B) WDL5 transgenic seedlings exhibit shorter hypocotyls after growth on MS in the dark for 5 days. The graph shows the average hypocotyl length measured from at least 38 seedlings under dark growth conditions. ttest, **P < 0.01. Error bars represent the mean ± SD. Supplemental Figure S8. WDL5 May Not Be Involved in Ethylene-Promoting Hypocotyl Cell Elongation in the Light. 2

(A) WDL5 expression levels were determined using quantitative real-time PCR with RNA purified from light-grown wildtype, ein2-5 and EIN3 overexpression seedlings. Error bars represent ± SD (n = 3). The wdl5-1 mutant shows similar hypocotyl lengths as the wildtype when grown on MS for 7 days in the absence (B) and presence of 10 M ACC (C). (D) The graph shows the hypocotyl length measured from at least 40 seedlings under light growth conditions. Error bars represent ± SD

3

4