Plant Physiology Preview. Published on September 5, 2017, as DOI:10.1104/pp.16.01764

1

A histone code reader and a transcriptional activator interact to regulate genes

2

for salt tolerance

3 4

Wei Wei1, Jian-Jun Tao1, Hao-Wei Chen1, Qing-Tian Li1, 2, Wan-Ke Zhang1, Biao

5

Ma1, Qing Lin1, Jin-Song Zhang1, 2, *, Shou-Yi Chen1,*

6 7

Author contribution

8

W. W., performed experiments and drafted the initial manuscript; J.-J.T., H.-W.C.,

9

Q.-T.L., assistance in some experiments; W.-K.Z., B.M., Q.L., data analysis; J.-S.Z.,

10

S.-Y.C., conceived projects, designed experiments and wrote the paper.

11 12

Funding information

13

This work is supported by the National Key Research and Development Program of

14

China (2016YFD0100304), 973 projects (2015CB755702), Transgenic research

15

project (2016ZX08004002-003 and 2016ZX08009003-002), National Natural Science

16

Foundation of China (31530004), the Strategic Priority Research Program of the

17

Chinese Academy of Sciences (XDA08020106) and State Key Lab of Plant

18

Genomics.

19 20 21 22

1

State Key Lab of Plant Genomics, Institute of Genetics and Developmental Biology,

Chinese Academy of Sciences, Beijing 100101, China 2

University of Chinese Academy of Sciences, Beijing 100049, China

23

Address: Chaoyang District, Beichen West Road, Campus #1, No 2, Beijing 100101,

24

China

25 26

Email: Wei Wei ([email protected]), Jian-Jun Tao ([email protected]),

27

Hao-Wei Chen ([email protected]), Qing-Tian Li ([email protected]),

28

Wan-Ke Zhang ([email protected]), Biao Ma ([email protected]), Qing

29

Lin ([email protected]) 1 Downloaded from on September 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.

Copyright 2017 by the American Society of Plant Biologists

30 31

*Corresponding author: Shou-Yi Chen ([email protected]) or Jin-Song Zhang

32

([email protected]),

33

8610-64806711

Tel:

8610-64806623

or

8610-64807601,

Fax:

34 35

Short title: PHD6/LHP1 complex confers stress tolerance

36 37

Total word count (without cover page, References and Supporting Information): 7052

38 39

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

40

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

41

authors

42

([email protected]).

are

Jin-Song

Zhang

([email protected])

and

Shou-Yi

43 44 45 46 47 48 49 50 51 52 53 54

2 Downloaded from on September 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.

Chen

55

Abstract

56

Plant homeodomain (PHD) finger proteins are involved in various developmental

57

processes and stress responses. They recognize and bind to epigenetically modified

58

histone H3 ‘tail’ and function as ‘histone code readers’. Here we report that GmPHD6

59

reads low methylated histone H3K4me0/1/2 but not H3K4me3 with its N-terminal

60

domain instead of the PHD finger. GmPHD6 does not possess transcriptional

61

regulatory ability but has DNA-binding ability. Through the PHD finger, GmPHD6

62

interacts with its co-activator, LHP1-1/2 to form a transcriptional activation complex.

63

Using a transgenic hairy root system, we demonstrate that over-expression of

64

GmPHD6 improves stress tolerance in soybean plants. Knocking down the LHP1

65

expression disrupts this role of GmPHD6, indicating that GmPHD6 requires LHP1

66

functions during stress response. GmPHD6 influences expression of dozens of

67

stress-related genes. Among these, we identified three targets of GmPHD6, including

68

ASR (ABA-stress-ripening induced), CYP75B1 and CYP82C4. Overexpression of

69

each gene confers stress tolerance in soybean plants. GmPHD6 is recruited to

70

H3K4me0/1/2 marks and recognizes the G-rich elements in target gene promoters，

71

while LHP1 activates expression of these targets. Our study reveals a mechanism

72

involving two partners in a complex. Manipulation of the genes in this pathway

73

should improve stress tolerance in soybean or other legumes/crops.

74 75 76 77

Key Word: Plant homeodomain, histone code, H3K4me0/1/2, LHP1, soybean, abiotic

78

stress, co-activator

79 80 81 82 83 3 Downloaded from on September 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.

84

Introduction

85

The plant homeodomain (also called PHD finger) refers to a zinc-binding domain

86

of 50-80 amino acids (Bienz, 2006). PHD finger contains eight conserved

87

metal-binding residues, Cys4HisCys3. The eight residues are divided into four pairs

88

and are connected by variable sequence. Two zinc atoms are anchored in a

89

cross-braced manner; the first zinc atom is anchored by the first and third pairs,

90

whereas the second zinc atom is anchored by the second and the fourth pairs (Kwan et

91

al., 2003).

92

PHD fingers are usually involved in protein-protein interaction. Normally, their

93

binding

partners

are

epigenetically

modified

histone-proteins.

94

post-translational modifications (PTMs) of histones throughout the genome constitute

95

complicated ‘histone code’. For an example, histone H3 has different types of PTMs,

96

such as methylation, acetylation and phosphorylation etc. (Jenuwein and Allis, 2001).

97

Different PTMs lead to the difference in transcriptional states; methylated lysine4 (K4)

98

and K36 are associated with ‘on’ state, whereas methylated K9 and K27 are

99

associated with ‘off’ state (Jenuwein and Allis, 2001). The histone code is ‘readable’

100

and PHD finger is one of these code readers (Mellor, 2006). PHD fingers can

101

recognize different PTMs with sequence-dependent manner, especially the

102

methylation of lysine (Sanchez and Zhou, 2011). For example, PHD finger of AIRE1

103

reads H3K4me0 (unmethylated H3) (Org et al., 2008); GmPHD5 reads H3K4me2

104

(dimethylated H3K4) (Wu et al., 2011); Alfin1-like proteins in Arabidopsis prefer tri-

105

or di-methylated H3K4 (H3K4me3/2) (Lee et al., 2009); ING (inhibitor of growth)

106

group proteins bind to H3K4me3 (Pena et al., 2006); PHD1 of CHD4 is reader of both

107

H3K4me0 and H3K9me3, whereas PHD2 has the preference of H3K9me3 (Mansfield

108

et al., 2011). Even though several PHD fingers read the same code, they may result in

109

different regulatory mechanisms based on the characters of these readers. Two ING

110

group protein, ING2 and ING4, bind to H3K4me3. ING4 mediates crosstalk between

111

tri-methylation and acetylation of H3 and leads to activation of target genes (Hung et

112

al., 2009); however, ING2 interacts with H3K4me3 to ‘switch’ off the active genes

113

(Shi et al., 2006). 4 Downloaded from on September 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.

Various

114

PHD finger proteins are ubiquitous in eukaryotes. In plants, they are involved in

115

several important biological processes, such as meiosis, vernalization, anther

116

development and apical meristem maintenance (Wilson et al., 2001; Makaroff et al.,

117

2003; Amasino et al., 2006; Komeda et al., 2008). Among the plant PHD finger

118

proteins, there is an Alfin1 group. This group is specific in plants and only several

119

members are present in various plant species (Lee et al., 2009). Alfin1-like proteins

120

locate in nucleus, they have transcriptional repression ability and bind to the G-rich

121

(GTGGNG/GNGGTG) elements in the promoter regions of their target genes.

122

Previously, we have characterized the six Alfin1-like proteins in soybean and find that

123

the N-terminal domain plays a major role in DNA binding. GmPHD1 to GmPHD5

124

have transcriptional suppression activity but GmPHD6 does not have this activity

125

(Wei et al., 2009). The Alfin1-like proteins are usually associated with abiotic stress

126

responses (Wu et al., 2011; Song et al., 2013; Wei et al., 2015). They are also involved

127

in elongation of root hairs during phosphate starvation and seed germination process

128

(Chandrika et al., 2013; Molitor et al., 2014).

129

In the Alfin1 group, there are several special cases. For instance, Alfin1 found in

130

Medicago sativa was reported to bind to the promoter of MsPRP2 and enhance but

131

not suppress the expression of MsPRP2 (Bastola et al., 1998; Winicov and Bastola,

132

1999). Besides, AtAL6 from Arabidopsis is unable to bind to the G-rich element,

133

because the N-terminal has two crucial point mutations (Wei et al., 2015). In this

134

study, we reported another special case of Alfin1-like protein, GmPHD6, in soybean,

135

whose transcriptional regulatory ability is somehow missing. The N-terminal domain

136

of this protein can recognize the H3K4me0/1/2 and bind to the promoter of the target

137

gene, whereas its PHD domain can recruit a co-activator LHP1-1 or LHP1-2 for

138

transcriptional regulation. This protein complex would activate expression of

139

downstream genes for stress tolerance in soybean. Our study reveals a specific

140

mechanism for Alfin1-like proteins without direct transcriptional activation ability in

141

response to abiotic stress in plants.

142

5 Downloaded from on September 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.

143

6 Downloaded from on September 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.

144

Results

145

Over-expression of GmPHD6 improves salt stress tolerance in transgenic soybean

146

composites

147

Soybean has six Alfin1-like proteins named as GmPHDs. Five of these (GmPHD1

148

to GmPHD5) has transcriptional suppression activity and GmPHD2 improves stress

149

tolerance. GmPHD6 does not have such transcriptional suppression activity; however,

150

this protein can interact with other GmPHDs except GmPHD2, and the GmPHD6

151

gene can be induced by stresses (Wei et al., 2009). The GmPHD6 protein was pursued

152

for its possible roles in stress response and the mechanism involved was further

153

investigated. An Agrobacterium rhizogenes-mediated hairy root transformation

154

system

155

GmPHD6-overexpression (G6-OE) and GmPHD6-RNA interference (G6-RNAi)

156

constructs were injected into the hypocotyl of soybean seedlings for generation of

157

transgenic hairy roots (Fig. 1A). The transgenic hairy roots grew out of the infection

158

sites about two weeks later and the original roots were removed before subjecting to

159

stress treatment.

was

first

established,

and

the

K599

strain

harboring

the

160

Compared to control (K599) hairy roots, G6-OE roots had 45-fold increase of

161

GmPHD6 expression, whereas G6-RNAi roots had about 80% reduction of GmPHD6

162

expression (Fig. 1B). To determine the performance of these hairy roots, relative

163

growth rates (RGR) were measured and compared. After salt stress with 100 mM

164

NaCl, the RGR of G6-OE hairy roots was apparently higher whereas this parameter in

165

G6-RNAi roots was significantly lower than that in the control roots (Fig. 1C). The

166

leaves of the plants with G6-OE roots also performed better than the control leaves;

167

however, the leaves from the plants with G6-RNAi roots were more wilted than the

168

control ones (Fig. 1D, E). Relative electrolyte leakage was then measured to evaluate

169

membrane damage of these leaves. After salt stress, leaves from plants with G6-OE

170

roots showed lower leakage whereas the leaves from plants with G6-RNAi roots had

171

higher leakage compared to control plants (Fig. 1F). These results indicate that

172

GmPHD6 confers salt stress tolerance in soybean.

173 7 Downloaded from on September 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.

174

GmPHD6 interacts with LHP1-1 or LHP1-2 to activate gene expression

175

GmPHD6 has DNA binding ability and improves salt stress tolerance (Wei et al.,

176

2009; Fig. 1); however, it does not have transcriptional activation and/or suppression

177

ability (Wei et al., 2009). It is possible that GmPHD6 may recruit some co-factors for

178

regulation of downstream genes and affect stress responses. We thus screened the

179

possible proteins interacted with GmPHD6 by yeast two-hybrid assay, and two LHP1

180

proteins (like heterochromatin protein 1), namely LHP1-1 and LHP1-2, were

181

identified. The interaction of full-length LHP1 and GmPHD6 was revealed by blue

182

colors in yeast cells harboring AD-LHP1 and BD-GmPHD6 on QDO/X/A plates (Fig.

183

2A). The two LHP1 proteins share about 85% identity at the amino acid level. Both 8 Downloaded from on September 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.

184

LHP1-1 and LHP1-2 contain a chromo domain (chromatin organization modifier) and

185

a chromo shadow (CSD) domain in the C-terminal (Fig. 2B). Organ-specific

186

expression was analyzed, and GmPHD6, LHP1-1 and LHP1-2 had similar expression

187

levels in roots and shoots/stems, while GmPHD6 was expressed higher than the two

188

LHP1 genes in leaves and flowers (Fig. S1).

189

To confirm the interaction, we incubated GST or GST-GmPHD6 and MBP-LHP1-1

190

or MBP-LHP1-2 with Glutathione-Sepharose 4B beads to perform a GST pull-down

191

assay. GST-GmPHD6 but not GST could pull down MBP-LHP1-1 or MBP-LHP1-2

192

(Fig. 2C). Another soybean PHD protein, GmPHD5, could not pull down any LHP1

193

protein (Fig. 2C). These results indicate that GmPHD6 could specifically interact with

194

LHP1-1 or LHP1-2 in vitro. The in vivo interaction was further confirmed by the

195

co-immunoprecipitation (Co-IP) assay. Flag-GmPHD6 and MYC-LHP1 were

196

transiently expressed in tobacco leaves for the IP assay. Protein extract was incubated

197

with agarose beads that crosslinked with anti-c-MYC antibody. A western blotting

198

assay showed that these agarose beads captured c-Myc-LHP1-1 and Flag-GmPHD6.

199

When empty MYC vector and Flag-GmPHD6 were expressed, the agarose beads

9 Downloaded from on September 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.

200

could not capture any protein (Fig. 2D). This result indicates that GmPHD6 interacted

201

with LHP1 in vivo.

202

A transcriptional activation assay was performed to test whether LHP1-1 and

203

LHP1-2 had any transcriptional regulatory effect. In a dual-luciferase reporter (DLR)

204

assay system, a firefly luciferase (LUC) was used as a reporter and a Renilla

205

luciferase was used as an internal control. The LUC gene was driven by a minimal

206

35S promoter connected by five copies of the GAL4 binding element (Fig. S2A).

207

GAL4 DNA binding domain (BD) could bind to the GAL4 element. If the tested

208

protein has transcriptional activation activity, the BD fusion protein will enhance the

209

expression of LUC gene and thus have a higher relative LUC activity than the control

210

(BD). The result showed that LHP1-1 and LHP1-2 but not GmPHD6 had

211

transcriptional activation ability (Fig. 2E). We further tested the transcriptional

212

regulatory effect of the GmPHD6/LHP1 complex using a modified assay.

213

BD-GmPHD6 and 35S-LHP1 were co-expressed in Arabidopsis protoplasts (Fig.

214

S2B). DLR assay showed that BD-GmPHD6 plus LHP1-1 or LHP1-2 had higher

215

LUC activity than control samples (BD + LHP1-1 or BD + LHP1-2 or BD-GmPHD6

216

+ Actin) (Fig. 2F and Fig. S2C). These results indicate that BD-GmPHD6 formed a

217

complex with LHP1-1 or LHP1-2 to bind to the GAL4 element through BD-GmPHD6

218

and to activate the expression of LUC gene through LHP1-1 or LHP1-2 recruited to

219

GmPHD6.

220 221

Domain analysis of GmPHD6 binding to LHP1, DNA element and histone H3

222

GmPHD6, like other GmPHD proteins, consists of two conserved domain, the

223

N-terminal domain (N) and the PHD finger, connected by a variable region (V) (Fig.

224

3A). Since GmPHD6 interacted with LHP1-1 or LHP1-2 (Fig. 2), we investigated

225

which domain in GmPHD6 is responsible for the interaction. Each of the single

226

domain including G6N, G6V and G6PHD (G6P), or combination of them (G6NV,

227

G6VP) was expressed as GST-tagged proteins and tested in GST pull-down assay. The

228

results showed that G6P and G6VP could pull down the LHP1-1 in vitro, whereas

229

other domains or combinations could not (Fig. 3A, lower panel), indicating that the 10 Downloaded from on September 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.

230

PHD finger plays a major role in protein interaction.

231

Our previous study shows that the N-terminal domains of other GmPHD proteins

232

play major roles in DNA binding (Wei et al., 2009), and mutation of two amino acids

233

in the N-terminal domain caused the functional loss of G-rich element binding activity

234

in Arabidopsis PHD protein AtAL6 (Wei et al., 2015). Thus, we performed an EMSA

235

assay to test the DNA binding property of GmPHD6. The results showed that full

236

length of GmPHD6 and the N-terminal domain bound to the G-rich (GTGGAG)

237

elements, whereas the mutated N-terminal domain G6NM (V34M, E35V) could not

238

bind to the G-rich elements (Fig. 3B). These results indicate that the N-terminal

239

domain of GmPHD6 is responsible for DNA binding.

240

Because the PHD domain often interacts with the histone code in chromatin, we

241

tested whether GmPHD6 and LHP1-1 or LHP1-2 could associate with histone H3. A

242

peptide (biotin conjugated H3 peptide) pull-down assay was performed and the results

243

showed that GmPHD6 could bind to unmethylated (H3), monomethylated (H3K4me)

244

and dimethylated H3 at K4 (H3K4me2) but not the trimethylated H3 at K4

245

(H3K4me3) (Fig. 3C). It should be noted that LHP1-1 and LHP1-2 showed very weak

246

binding intensity towards H3, H3K4me, and H3K4me2 compared to the strong input 11 Downloaded from on September 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.

247

signals (Fig. 3C). All these results suggest that GmPHD6, but not LHP1, played major

248

roles in H3K4me0/1/2 recognition.

249

examined for their interaction with H3 peptide. It is shown that G6N and G6NV, but

250

not G6P or G6VP, could bind to H3K4me0/1/2 (Fig. 3D). Only very weak interaction

251

with the H3K4me was noted when single V domain was studied (Fig. 3D). These

252

results indicate that N-terminal domain of GmPHD6 may play a major role in ‘histone

253

code’ reading in addition to its DNA binding.

Different domains of GmPHD6 were further

254 255

LHP1 is required for salt stress tolerance

256

Since the GmPHD6 confers stress tolerance and interacts with LHP1-1 and LHP1-2,

257

we studied whether the LHP1-1 and LHP1-2 play any roles in salt stress responses.

258

We overexpressed LHP1-1 or LHP1-2 gene in transgenic hairy roots of soybean

259

plants and tested their performance. We found that overexpression of LHP1-1 or

260

LHP1-2 could not enhance stress tolerance of the transgenic hairy roots (Fig. S3). We

261

then selected a 379 bp fragment of LHP1-1 coding region for RNA interference test.

262

This fragment showed 97% identity with that from LHP1-2 (Fig. S4). The expression

263

of LHP1-1 and LHP1-2 in LHP-RNAi hairy roots was simultaneously knocked down

264

to less than 10% of that in control roots (K599) (Fig. 4A). After salt stress for three

265

days, the LHP-RNAi transgenic hairy roots grew shorter than control roots; the

266

relative growth rate of LHP-RNAi hairy roots was significantly reduced compared to

267

the value in control roots (Fig. 4B and C). Meanwhile, leaves of soybean plants with

268

LHP-RNAi transgenic roots had more severe damage and higher electrolyte leakage

269

than that of control plants (Fig. 4D, E and F). The results demonstrate that decrease of

270

LHP1 gene expression leads to sensitive responses in soybean seedlings, indicating

271

that LHP1 is required for plant tolerance to salt stress.

272 273

LHP1 acts downstream of GmPHD6 for salt tolerance

274

Considering that overexpression of the LPH1 could not enhance stress tolerance,

275

and GmPHD6 formed complex with LPH1, we tested whether co-overexpression of

276

these two components in transgenic hairy roots would have any effects on stress 12 Downloaded from on September 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.

277

response.

We

mixed

equal

amounts

of

278

GmPHD6-overexpression construct and LHP1-1-overexpression construct and

279

infected soybean hypocotyls for generation of co-expressed transgenic hairy roots.

280

The soybean plants with both GmPHD6- and LHP1-1-overexpressing transgenic hairy

281

roots showed better salt tolerance than the plants with single GmPHD6-transgenic

282

roots or control roots, as judged from the leaf performance and relative electrolyte

283

leakage (Fig. 5A, B and C). The expression of GmPHD6 and LHP1-1 was higher in

284

co-transgenic hairy roots than that of K599 control (Fig. 5D). However, the GmPHD6

A.

rhizogenes

13 Downloaded from on September 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.

harboring

285

level was lower in the co-transgenic roots than the single GmPHD6-trangenic roots

286

(Fig. 5D). These results likely indicate that the cooperation of GmPHD6 and LHP1 in

287

a complex may be important for salt tolerance.

288

To study the genetic relationship between GmPHD6 and LHP1, equal amounts of A.

289

rhizogenes harboring GmPHD6-overexpression construct or LHP1-RNAi construct

290

were mixed and co-injected to soybean seedlings for infection and generation of

291

transgenic hairy roots. After salt treatment for three days, the soybean seedlings with

292

the co-transgenic hairy roots had leaves more damaged than those from control plants. 14 Downloaded from on September 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.

293

The electrolyte leakage was also higher in leaves of co-transgenic composites than

294

that of control plants (Fig. 5E, F and G). The expression of GmPHD6 was much

295

higher in co-transgenic hairy roots than that of control roots; however, the expression

296

of LHP1-1 and LHP1-2 in co-transgenic hairy roots was significantly reduced

297

compared to that in the control roots (Fig. 5H). These results demonstrate that even

298

though GmPHD6 was highly expressed, which is beneficial for salt tolerance, the

299

knock-down of the two LHP1 genes would disrupt the salt tolerance, suggesting that

300

GmPHD6 depends on LHP1; or LHP1 acts downstream of GmPHD6 for stress

301

tolerant response in soybean.

302 303

Two conserved residues at the N-terminal domain is necessary for

304

GmPHD6-mediated salt tolerance

305

The mutation of two amino acids in the N-terminal domain caused the functional

306

loss of G-rich element binding activity in GmPHD6 (Fig. 3B). We then tested whether

307

mutation of the two conserved corresponding amino acids would affect GmPHD6

308

function. A mutated form of GmPHD6, G6M (V34M, E35V), was generated and

309

transformed into soybean hairy roots (Fig. 6A). The soybean plants with the

310

transgenic hairy roots grew worse than control plants in 100 mM NaCl (Fig. 6B and

311

C). Meanwhile, the electrolyte leakage in leaf of soybean seedlings with G6M-OE

312

transgenic roots was significantly higher than that of control plants (Fig. 6D). The

313

transgenic hairy roots had very high expression of this mutated gene (about 200 fold)

314

(Fig. 6E). These results indicate that over-expression of this mutated GmPHD6 gene

315

caused salt sensitivity in soybean plants.

316 317

GmPHD6 specifically regulates downstream genes for salt stress tolerance

318

To identify the downstream genes that regulated by GmPHD6, an RNA sequencing

319

analysis was performed. RNA extracted from G6-OE, G6-RNAi and K599 control

320

hairy roots was used for RNA sequencing. Data of Clean reads in RNA sequencing is

321

shown in Table S1. Compared to the transcripts of K599 control roots, G6-OE roots

322

had 553 up-regulated genes and 987 down-regulated genes, whereas G6-RNAi roots 15 Downloaded from on September 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.

323

had 2707 up-regulated genes and 3032 down-regulated genes (Fig. S5A). We focused

324

on the downstream genes that were up-regulated in G6-OE roots and down-regulated

325

in G6-RNAi root samples. A venn diagram showed that there were 59 of these

326

downstream genes (Fig. S5B and Table S3). We further tested thirteen of them using

327

quantitative RT-PCR. These genes were all up-regulated in G6-OE roots and

328

down-regulated in G6-RNAi roots (Fig. 7A and Fig. S5C). The expression of target

329

genes depends on the binding of GmPHD6 towards their promoter regions and the

330

activation effect of LHP1-1 or LHP1-2. These target genes should not be up-regulated

331

in G6M-OE root samples and should be down-regulated in LHP1-RNAi samples.

332

Based on this assumption, four genes out of the thirteen, namely CYP75B1,

333

CYP71A22, ASR and CYP82C4, were found to have the consistent expression pattern

334

(Fig. 7A).

335

We transformed the CYP75B1, ASR and CYP82C4 into soybean hairy roots (Fig.

336

7B) to test gene functions in response to salt stress. After salt treatment for three days, 16 Downloaded from on September 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.

337

the soybean seedlings with transgenic hairy roots overexpressing the three genes grew

338

better and had higher relative growth rate than control roots (Fig. 7C and D). The

339

aerial part of the transgenic composites also had better performance and their leaves

340

had lower electrolyte leakage compared to control plants (Fig. 7C and E). These

341

results indicate that the downstream genes play positive roles in salt tolerance of

342

soybean.

17 Downloaded from on September 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.

343

Through

sequence

scanning,

we

found

several

G-rich

elements

344

(GTGGNG/GNGGTG) in promoter regions of the downstream genes, except for

345

CYP75B1. A promoter LUC assay was performed to test whether the GmPHD6 could

346

bind to these promoter regions. The promoter regions of the four downstream genes

347

were cloned and inserted in front of LUC gene, replacing the original GAL4 elements

348

and the mini 35S promoter in the GAL4-LUC reporter. A GmPHD6-VP16 fusion

349

construct was used as an effector (Fig. S2D). If GmPHD6 can bind to the promoter

350

region, the expression of LUC gene would be activated and up-regulated by VP16.

351

The result showed that the promoter activity of the CYP71A22, ASR and CYP82C4

352

but not CYP75B1 was enhanced, suggesting that GmPHD6 most likely can bind to the

353

promoters of CYP71A22, ASR and CYP82C4 (Fig. 7F left).

354

We further examined the DNA binding ability of the control fusions by replacing

355

GmPHD6 with AtActin7, BD or G6M in the GmPHD6-VP16 construct (Fig. S2D).

356

These new constructs were used as negative controls for G-rich element binding since

357

Actin does not bind to DNA, and BD domain and the mutated GmPHD6 protein G6M

358

(V34M, E35V) cannot bind to G-rich elements. In the presence of PCYP82C4-LUC

359

reporter, Actin-VP16 and BD-VP16 produced similar low LUC activity as the control

360

sample (LUC + G6-VP16) (Fig. 7F, right panel). The mutated GmPHD6 protein,

361

G6M, generated low LUC activity either. In contrast, the combination of

362

PCYP82C4-LUC plus GmPHD6-VP16 produced higher LUC activity compared to the

363

above negative controls (Fig. 7F right). These results indicate that GmPHD6

364

specifically binds to the promoter region of CYP71A22, ASR and CYP82C4 for

365

regulation of gene expression.

366 367

GmPHD6 promotes stomatal closure upon salt treatment

368

Because the aerial parts of the soybean plants with GmPHD6-overexpressing hairy

369

roots also showed stress tolerant phenotype (Fig. 1 and 5), we further examined the

370

possible mechanisms involved. We found that the stomata opening was significantly

371

reduced in leaves of soybean plants with GmPHD6-overexpressing hairy roots

372

compared to the control plants after salt stress for 1 h (Fig. 8A and B). In contrast, the 18 Downloaded from on September 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.

373

stomatal aperture was increased in leaves of soybean plants with GmPHD6-RNAi

374

roots (Fig. 8A and B). We also measured relative water content (RWC) in all the

375

tri-foliate leaves after the plants with transgenic hairy roots were stressed in 100 mM

376

NaCl for three days.. Leaves from plants with GmPHD6-overexpressing hairy roots

377

had higher RWC than that of control plants, whereas leaves from plants with RNAi

378

hairy roots had lower RWC (Fig. 8C). These results indicate that GmPHD6 from

379

transgenic hairy roots may improve stress tolerance of aerial parts through regulation

380

of stomata closing and water contents in leaves.

381

Usually, the stomata closure is related to ABA pathway. However, GmPHD6 did

382

not regulate ABA-related genes (Fig. S6A). We further analyzed the transcriptome

383

data and found several homologues of CCD (carotenoid cleavage dioxygenase) genes,

384

which take part in strigolactone biosynthesis (Lopez-Raez et al., 2010). Their

385

expressions had about 1.5 fold increases in GmPHD6-overexpressing hairy roots and

386

more than 80% reduction in RNAi hairy roots (Fig. S6B). These expression patterns

387

were further confirmed by qRT-PCR (Fig. 8D). Considering that the strigolactone can

388

be transported from roots to shoots, and it is also involved in stomata closure during 19 Downloaded from on September 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.

389

stress response (Ha et al., 2014), we propose that overexpressing GmPHD6 in hairy

390

roots may confer salt tolerance in the aerial part of the soybean plants at least partially

391

through strigolactone-mediated stomata closure.

392 393

20 Downloaded from on September 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.

394

Discussion

395

Typically, a transcription factor/regulator possesses two functional domains, DNA

396

binding domain and transcriptional regulatory domain. The regulatory domain has

397

either transcriptional activation or suppression activity. GmPHD6 belongs to a small

398

Alfin1-like group of PHD finger proteins. Normally, Alfin1-like proteins are

399

transcriptional suppressors; they play biological roles through inhibition of their

400

downstream genes (Wei et al., 2009; Wei et al., 2015). However, GmPHD6 somehow

401

loses its suppression activity (Fig. 2E). It is rationally presumed that GmPHD6 may

402

recruit certain transcriptional co-factor, as in the case of RVE4/8 (REVEILLE), which

403

interacts

404

CLOCK-REGULATED) for regulation of downstream genes (Xie et al., 2014). Using

405

yeast two-hybrid system, we identified two chromo domain-containing transcriptional

406

activators, LHP1-1 and LHP1-2, which specifically interacted with GmPHD6 but not

407

GmPHD5 (Fig. 2). GmPHD6 may recruit LHP1-1/2 through its PHD domain to form

408

a transcriptional regulatory complex, with N-terminal domain of GmPHD6 acting as a

409

DNA binding domain and LHP1-1/2 acting as a transcription-activating domain (Fig.

410

9).

411

with

GmPHD6

co-activators

enhanced

LNK1/2

stress

(NIGHT

tolerance

in

LIGHT–INDUCIBLE

soybean

plants

AND

with

412

GmPHD6-overexpressing transgenic hairy roots. This function requires the DNA

413

binding ability of GmPHD6 N-terminal domain since mutation of the two conserved

414

residues in the N-terminal caused the inability of GmPHD6 to bind to the promoter of

415

its downstream genes (Fig. 7F). The mutation version of the GmPHD6 even led to salt

416

sensitivity when overexpressed in transgenic hairy roots (Fig. 6), possibly due to the

417

formation of a non-functional complex, where the mutated GmPHD6 may compete

418

with and replace the original GmPHD6 for LHP1 association and further affect stress

419

response.

420

Although the LHP1-1 and LHP1-2 associated with GmPHD6, over-expression of

421

LHP1-1 and LHP1-2 could not enhance stress tolerance in transgenic hairy roots (Fig.

422

S3). However, the two LHP1 proteins are required for stress tolerance as judged from

423

the fact that soybean seedlings with LHP1-RNAi transgenic roots are sensitive to salt 21 Downloaded from on September 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.

424

stress (Fig. 4). It is the GmPHD6/LHP1 complex that plays essential roles in salt

425

tolerance, and hence co-transformation of the GmPHD6 and LHP1-1 confers even

426

higher salt tolerance than overexpressing GmPHD6 alone (Fig. 5). Since there are two

427

LHP1 proteins in soybean, sharing a high similarity of about 85% (Fig. S4), LHP1

428

proteins may be excessive compared to GmPHD6 in roots. Consistently, the total

429

expression level of LHP1 (LHP1-1 plus LHP1-2) is about twice of that of GmPHD6

430

in roots (Fig. S1). Without enough amount of GmPHD6, the overexpressed LHP1

431

proteins would be unbounded and thus ineffective. Moreover, knocking down the

432

LHP1-1/2 expression in the background of GmPHD6 overexpression abolished stress

433

tolerance in soybean, demonstrating that GmPHD6 requires the LHP1-1/2 for stress

434

tolerance (Fig. 5). Since GmPHD6 can bind to G-rich element but have no

435

transcriptional regulatory activity, it is reasonable that GmPHD6recruits LHP1-1/2 for

436

transcriptional activation of downstream genes in response to different abiotic

437

stresses.

438

Previous studies find that the PHD finger could ‘read’ histone code, especially

439

trimethylated K4 of histone H3 (H3K4me3) (Li et al., 2006; Pena et al., 2006). In the

440

present study, GmPHD6 also binds to methylated histone tails, but with a different

441

manner. GmPHD6 could bind to H3K4me0/1/2, but not H3K4me3 (Fig. 3C). It is

442

interesting to note that the N-terminal domain but not the PHD finger of GmPHD6

443

reads histone code (Fig. 3D). The functions of PHD fingers diverge even in the Alfin1

444

group. For example, the PHD finger of AtAL1, AtAL4 and AtAL7 could bind to

445

H3K4me2/3, but the PHD finger of AtAL3 did not have this ability (Lee et al., 2009).

446

Meanwhile, another soybean PHD protein, GmPHD5, has the preference of 22 Downloaded from on September 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.

447

recognizing H3K4me2 (Wu et al., 2011). These differences may be caused by the

448

sequence diversity in PHD fingers. It is also reported that, the binding partners of a

449

PHD finger were not necessarily histone proteins; they could be non-histone proteins

450

as well (Li and Li, 2012). Just like the present case of GmPHD6, the PHD finger

451

recognized LHP1 as its binding partner (Fig. 3A). Interestingly, while the N-terminal

452

domain of GmPHD6 can bind to histone H3 (Fig. 3D), this domain also binds to DNA

453

element (Fig. 3B). The N-terminal domain, consisting of about 120 amino acids, is

454

named according to the sequence similarity among Alfin1 family proteins. It is not

455

clear why the N-terminal domain of GmPHD6 can bind to both the histone

456

H3K4me0/1/2 and DNA elements. It is possible that this domain may contain two

457

functional sub-domains, with the former part for DNA binding and the latter part for

458

H3 binding. The fact that mutation of the residues at 34th and 35th disrupted DNA

459

binding of N-terminal domain may support the above prediction (Fig. 3B). Further

460

studies should be conducted to test such a possibility.

461

LHP1 consists of two evolutionarily conserved domains, the chromo domain (CD)

462

near the N-terminal and the chromo shadow domain (CSD) at the C-terminal (Fig.

463

2B). The CD is known as a methylated histone H3 binding module. Generally, LHP1

464

is known to bind to transcriptional repressive mark of H3K9me3 (Stewart et al., 2005).

465

In Arabidopsis, AtLHP1 regulates development through repression of its target genes

466

(Cui and Benfey, 2009; Exner et al., 2009). Here we report different functions of two

467

soybean LHP1 proteins in transcriptional activation (Fig. 2E and Fig. 3C). Except for

468

the two conserved domains, the two soybean LHP1s showed very little similarity with

469

AtLHP1. LHP1 generally has a hinge region (HR). The HRs are variable among

470

different LHP1 proteins, which may exert different biochemical functions, including

471

RNA binding and transcriptional activation (Hiragami and Festenstein, 2005;

472

Lomberk et al., 2006; Keller et al., 2012). Therefore, soybean LHP1-1 and LHP1-2

473

play completely different biochemical and biological roles from AtLHP1. Although

474

the CSD shares similarity with the CD, it may function differently as a dimerization

475

module (Mendez et al., 2011; Nishibuchi and Nakayama, 2014). Thus, it is possible

476

that the CSD may interact with the PHD finger of GmPHD6 to form the complex. 23 Downloaded from on September 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.

477

Compared to LHP1, GmPHD6 plays a major role in recognizing H3K4me0/1/2

478

(Fig. 3C). Histone H3K4me0/1/2 may recruit GmPHD6, which further recruits its

479

co-factor LHP1 to form a transcriptional regulatory complex (Fig. 9). GmPHD6 may

480

act as code reader since it not only ‘reads’ histone code but also ‘reads’ DNA

481

cis-element in the DNA strands (Fig. 3B, 3C and Fig. 7F). After GmPHD6 locates the

482

target genes, LHP1 activates expression of them (Fig. 2E and Fig. 9). Normally,

483

H3K4me3 is associated with active transcription (Mellor, 2006). However, GmPHD6

484

prefers low-methylated H3K4 (H3K4me0/1/2), but not H3K4me3 (Fig. 3C).

485

Generally, under normal growth condition, stress-related genes are not highly

486

expressed. They can be induced by different stresses and in various organs of plants. It

487

is reported that genes associated with H3K4me3 are highly expressed but low

488

tissue-specific, whereas those genes associated with H3K4me and H3K4me2 are

489

highly tissue-specific but not always activated in transcription (Zhang et al., 2009). It

490

is therefore likely that, in response to stresses, GmPHD6 may recognize

491

low-methylated H3K4 and interact with LHP1-1/2 to form a complex, which activates

492

the associated target genes for stress tolerance in a specific manner (Fig. 9). It should

493

be noted that while the H3K4me0/1/2-GmPHD6-LHP1 complex is mainly derived

494

from the transgenic root studies, the complex may also work similarly in aerial parts

495

of soybean plants although further investigation should be performed to corroborate

496

this prediction.

497

Unlike other Alfin1-like proteins suppressing expression of negative factors during

498

stress response (Wei et al., 2009; Wei et al., 2015), the interaction of GmPHD6 with

499

LHP1 activates transcription of its target genes (Fig. 7A and Fig. S5B). These target

500

genes play positive roles in stress tolerance (Fig. 7A and Table S2). Three of the four

501

possible targets are cytochrome P450-coding genes. Cytochrome P450 superfamily

502

consists

503

oxidation/reduction reactions of enormous organic chemicals (Munro et al., 2013;

504

Rendic and Guengerich, 2015). They are also involved in drug metabolism, endocrine

505

activity, steroid and hormone biosynthesis, and other processes (Girvan and Munro,

506

2016; Guengerich et al., 2016). In plants, CYP genes take part in the biosynthesis or

of

large

numbers

of

monooxygenase

enzymes,

which

24 Downloaded from on September 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.

catalyze

507

catabolism of several phytohormones, such as cytokinin, ABA, GAs (Gibberellins)

508

and BRs (Brassinosteroids) (Saito et al., 2004; Takei et al., 2004; Zhu et al., 2006;

509

Sakamoto et al., 2012; Yoshida, 2012). Recently, increasing evidence showed that

510

P450s confer abiotic stress tolerance in plants (Rai et al., 2015; Tamiru et al., 2015;

511

Wang et al., 2016). Presently, we identified three CYP genes (CYP75B1, CYP71A22

512

and CYP82C4) up-regulated by GmPHD6 (Fig. 7B). CYP75B1 encodes enzyme that

513

mediates synthesis of phenylpropanoid; CYP71A22 is associated with synthesis of

514

furanocoumarins; CYP82C4 is related to transport of iron ion (Rupasinghe et al., 2003;

515

Murgia et al., 2011; Chen et al., 2014). Overexpression of CYP82C4 and CYP75B1

516

enhance salt tolerance in soybean plants with transgenic hairy roots (Fig. 7). The

517

relevant regulatory mechanism requires further investigation.

518

ASR is a transcription factor with an ABA/WDS (ABA and water deficit stress

519

induced) domain at its C-terminal end. This protein is usually associated with ABA

520

transduction and fruit ripening (Jia et al., 2016). ASR genes are also stress-responsive

521

genes (Yang et al., 2005; Arenhart et al., 2013). A wheat ASR gene, TaASR1, was

522

reported to confer drought tolerance in transgenic tobacco through regulating ROS

523

signaling (Hu et al., 2013). Our present findings indicate that ASR is directly regulated

524

by the GmPHD6/LHP1 complex, and promotes salt tolerance in soybean (Fig. 7).

525

It is interesting to note that although the aerial parts of the soybean plants with

526

GmPHD6-overexpressing hairy roots were non-transgenic, they also exhibited salt

527

tolerant phenotype, most likely due to stomatal closure and higher water contents

528

maintained (Fig. 1 and 8). Usually ABA could promote stomatal closure and reduce

529

water loss (Acharya and Assmann, 2009). However, overexpressing GmPHD6 did not

530

enhance expression of ABA-related genes substantially (Fig. S6A). Instead, we found

531

expression of several CCD genes was up-regulated by GmPHD6 (Fig. S6B and Fig.

532

8D). Arabidopsis CCD7 and CCD8 take part in strigolactone synthesis (Lopez-Raez et

533

al., 2010), and strigolactones are root-derived hormones, which could be transported

534

from roots to the aerial parts of plants (Kohlen et al., 2011; Seto and Yamaguchi,

535

2014). Moreover, strigolactone plays important roles in stress tolerance through

536

mediating stomatal closure (Ha et al., 2014). Another CCD gene, CCD1 was also 25 Downloaded from on September 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.

537

regulated by GmPHD6 (Fig. 8). CCD1 is involved in the biosynthesis of

538

cyclohexenone but not strigolactone, yet CCD1 is also related to stress response (Sun

539

et al., 2008; Kim et al., 2014). Our findings suggest that overexpressing GmPHD6 in

540

hairy roots may affect strigolactone synthesis and enhance salt tolerance in the aerial

541

parts of soybean through promoting stomatal closure and maintaining water content.

542

Other factors may also be involved in the stress tolerance of the soybean aerial parts

543

(Pan et al., 2016).

544

Taken together, we propose a working model for the GmPHD6-LHP1-1/2

545

transcriptional activation complex (Fig. 9). In response to a given stress, GmPHD6 is

546

recruited to low methylated H3 (H3K4me0/1/2). Through the PHD finger, GmPHD6

547

associates with LHP1, probably at the CSD in the C-terminal end. GmPHD6 ‘reads’

548

the histone code and recognizes the G-rich elements in target gene promoter, whereas

549

LHP1 functions as a transcriptional activator. In this way, the two partners form a

550

transcriptional activation complex to activate target gene expression for stress

551

tolerance in soybean. Our study sheds light on the mechanism of Alfin1-like PHD

552

proteins in stress response. Manipulation of the complex and/or target genes may

553

improve abiotic stress tolerance in soybean and/or other legumes/crops.

554 555

Materials and Methods

556

Plant materials

557

Soybean cultivar Kefeng 1 (Glycine max, KF1) was used for hairy root

558

transformation. Seeds were germinated on vermiculate soil at 28ºC and grown for one

559

week before infection.

560 561

Yeast two-hybrid assay

562

Yeast two-hybrid assay was performed following the method described in

563

MatchmakerTM Gold yeast two-hybrid system (Clontech, http://www.clontech.com/).

564

The coding sequence of GmPHD6 was cloned into the BamH I/Sal I sites of pGBKT7

565

and the library of soybean cDNAs were inserted into the pGADT7 vector. The bait

566

vector was transfected into yeast strain Y2HGold and the library vector was 26 Downloaded from on September 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.

567

transfected into yeast strain Y187. After tested for auto-activation (BD-GmPHD6 has

568

no auto-activation activity) and toxicity, the bait strain was combined with the library

569

and slowly (50 rpm) shaken at 30°C for 20–24 hr. The mated yeast strains were grown

570

on QDO/X/A (SD/-Ade/-His/-Leu/-Trp/X-a-Gal/AbA) plates for three days and blue

571

colonies indicated positive interactions. Plasmids of these positive clones were

572

extracted and sequenced.

573 574

Agrobacterium rhizogenes-mediated transformation of soybean hairy roots and

575

stress treatments

576

For over-expression vector construction, the coding sequences of GmPHD6

577

(Glyma.09G068500), LHP1-1 (Glyma.16G079900) and LHP1-2 (Glyma.03G094200)

578

were

579

(http://www.arabidopsis.org/). For RNAi vector construction, the sense fragments of

580

GmPHD6, LHP1-1 and LHP1-2 were cloned into the Sac I/Kpn I sites of pZH01, and

581

the antisense fragments were cloned into the Xba I/Sal I sites. The hypocotyls of

582

one-week-old soybean seedlings (KF1) were injected with A. rhizogenes strain K599

583

harboring the pROKII or pZH01 construct following a method published in 2007

584

(Kereszt

585

35S-GmPHD6andLHP1-RNAi or 35S-GmPHD6 and 35S-LHP1-1 was mixed in

586

equal amount and injected to the hypocotyls of soybean seedlings. The infected

587

seedlings were kept in high humidity for about two weeks until the hairy roots were

588

generated from the wounded sites. The original roots were cut and the seedlings were

589

moved to water for recovery for three days. Seedlings with hairy roots of 2 to 10 cm

590

were transferred to 100 mM NaCl for 3 days as stress treatments. Leaf phenotypic

591

change was observed and root length was measured before and after the treatment.

592

The relative root growth rate was calculated to determine the performance of hairy

593

roots. Relative growth rate (%) = (average root elongation in stress) / (average root

594

elongation in water).Relative electrolyte leakage and relative water content were

595

measured to determine the performance of soybean leaves. After salt treatment, all the

596

tri-foliates were cut and dried at 60 °C for at least 2 d to measure water content.

cloned

et

into

al.,

the

2007).

BamH

For

I/Kpn

I

sites

co-transformation,

A.

of

pROKII

rhizogenes

27 Downloaded from on September 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.

vector

harboring

597

Relative water content (%) = (water content after salt treatment) / (water content in

598

water).

599 600

Quantitative RT PCR

601

To detect gene expression in transgenic hairy roots, we harvested transgenic hairy

602

roots from 10-15 plants for RNA extraction and gene expression analysis. A soybean

603

Tubulin fragment was used as an internal control.

604 605

Relative electrolyte leakage assay

606

After treatment in 100 mM NaCl for 3 d, the second trifoliate from the top was cut

607

for the relative electrolyte leakage assay. Soybean leaves were immersed in water,

608

vacuumed for 45 min and placed at room temperature for 2 h. Conductivity (K1) was

609

then measured. The leaves were then autoclaved for 15 min. The samples were shaken

610

at 200 rpm until water temperature dropped to room temperature. Conductivity (K2)

611

was measured again. Relative electrolyte leakage (%) = K1/K2.

612 613 614

Protein expression and GST or H3 peptide pull-down assay The coding regions for full length or various domains of GmPHD6 were inserted

615

into

the

BamH

I/Sal

I

sites

of

pGEX4T-1

vector

(GE

Healthcare,

616

http://www.gelifesciences.com/) to generate GST fusion proteins. The coding

617

sequences of LHP1-1 and LHP1-2 were cloned into the EcoR I/Sal I sites of

618

pMAL-c2x vector to generate MBP fusion proteins. The constructed plasmids were

619

transformed into the E. coli BL21 strain. GST fusion proteins were purified using

620

glutathione Sepharose 4B (GE Healthcare). MBP fusion proteins were purified using

621

amylase resin (New England).

622

For GST pull-down assay, GST fusion proteins were bound to glutathione

623

Sepharose 4B beads for 1h at room temperature in PBS buffer (137 mM NaCl, 2.7

624

mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4). MBP fusion proteins were then

625

added to the beads and incubated at 4 °C overnight. The beads were washed twice

626

with high-salt-washing buffer (25 mM Tris pH 8.0, 10% glycerol, 1 mM EDTA, 500 28 Downloaded from on September 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.

627

mM NaCl, 1 mM PMSF, 1 mM DTT, 1% Triton X-100) and six times with

628

low-salt-washing buffer (25 mM Tris pH 8.0, 10% glycerol, 1 mM EDTA, 150 mM

629

NaCl, 1 mM PMSF, 1 mM DTT, 1% Triton X-100). The beads were collected and

630

boiled with SDS loading buffer for 10 min. The eluted proteins were electrophoresed

631

on 4-20% SDS PAGE and transferred to a PVDF membrane (Millipore). Anti-GST or

632

Anti-MBP (Abmart) antibody was used as primary antibody and then goat anti-mouse

633

IgG-HRP (Abmart) was used as second antibody. Western blotting signal was detected

634

using a SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher).

635

For H3 peptide pull-down assay, GST or MBP or their fusion proteins were

636

incubated with 1 μg biotin conjugated H3 peptide (Millipore: 12-403 for H3, 12-563

637

for H3K4me, 12-460 for H3K4me2, 12-564 for H3K4me3) in binding buffer (50 mM

638

Tris pH 7.5, 300 mM NaCl, 0.1% NP-40) at 4 °C overnight. Dynabeads M-280

639

Streptavidin (Invitrogen) was then applied and incubated for 1h at 4 °C. The beads

640

was washed twice with high-salt-washing buffer and six times with low-salt-washing

641

buffer, as described above, and boiled with SDS loading buffer. Western blotting

642

described above was performed to detect the eluted proteins.

643 644

Co-immunoprecipitation (co-IP) assay

645

The coding sequence of GmPHD6 was cloned into pGWB412 vector to express

646

Flag-GmPHD6 protein. The coding sequence of LHP1-1 or LHP1-2 was cloned into

647

pGWB420 vector to express LHP1-MYC proteins. Plasmids were transformed into

648

Agrobacterium tumefaciens strain GV3101. A. tumefaciens harboring pGWB412,

649

pGWB420 and p19 (gene-silencing suppressor) were co-injected into tobacco leaves.

650

After growth at 25 °C for 3 d, tobacco leaves were harvested and ground in liquid

651

nitrogen to a fine powder. Nucleus was extracted with nuclei isolation buffer (0.25 M

652

sucrose, 15 mM PIPES pH 6.8, 5 mM MgCl2, 60 mM KCl, 15 mM NaCl, 1 mM

653

CaCl2, 0.9% Triton X-100, 1 mM PMSF) and lysed with nuclei lysis buffer (50 mM

654

HEPES pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1% sodium deoxycholate and 1%

655

Triton X-100) with protease and phosphatase inhibitors (Thermo Fisher). Co-IP assay

656

was performed using a c-MYC-Tag IP/Co-IP kit (Thermo Fisher) and detected by 29 Downloaded from on September 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.

657

western blotting.

658 659

DNA binding assay

660

Single-stranded complementary fragments containing 4X GTGGAG elements were

661

synthesized and labeled with biotin at their 5’-end (Invitrogen). Double-stranded

662

DNA probes were obtained by heating complementary fragments at 75ºC for 5min,

663

then slowly cooling in room temperature. The gel-shift assays were performed using

664

the LightShift Chemiluminescent EMSA Kit (Thermo Fisher Scientific).

665 666

Stomatal closure measurement

667

The soybean plants were kept in 100 mM NaCl for 1 h and the second tri-foliate

668

from the top were chosen for stoma observation. Soybean plants kept in water were

669

used as control samples.

670 671

Transcriptional activation assay

672

The transcriptional activation assay was performed using Arabidopsis protoplast

673

(Yoo et al., 2007) and Dual-Luciferase Reporter Assay System (Ohta et al., 2001). A

674

pUC19 vector containing five GAL4 binding elements and a firefly LUC gene was

675

used as reporter plasmid and pTRL vector including a Renilla LUC gene was used as

676

an internal control. For trans-activation assay of single gene, coding sequence of

677

GmPHD6, LHP1-1 and LHP1-2 were cloned into pRT-BD vector and a

678

pRT-BD-VP16 was used as a positive control. For trans-activation assay of

679

GmPHD6/LHP1 complex, LHP1-1 and LHP1-2 were cloned into pRT107 vector

680

driven by 35S promoter (kindly provided by Prof. Yan Guo, China Agricultural

681

University). For promoter-LUC assay, the GAL4 binding elements and TATA-box of

682

the reporter plasmid, in front of LUC gene, were replaced by the promoter regions of

683

downstream genes. The BD-coding sequence of pRT-BD-VP16 was replaced by

684

GmPHD6 to generate 35S-G6-VP16. A luminescence reader was used to detect LUC

685

signal (Promega, GloMax 20-20 luminometer).

686 30 Downloaded from on September 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.

687

RNA extracting and sequencing

688

Transgenic hairy roots were harvested for RNA extraction using TRNzol Reagent

689

(TIANGEN company). RNA-sequencing was performed by “1gene” company (1gene,

690

Hangzhou, China) using Illumina HiSeq. Each sample contained two replicates.

691

Adaptor sequence and low-quality reads were deleted and the clean reads were

692

mapped to soybean genome (https://phytozome.jgi.doe.gov/) using Tophat software

693

(http://ccb.jhu.edu/software/tophat/). Differentially expressed genes were analyzed

694

using

695

down-regulated transcripts, with fold change ≥2, were examined for possible

696

downstream

697

(http://bioinfogp.cnb.csic.es/tools/venny/index.html).

698

transcription PCR (RT-PCR) was performed to test the results of RNA-Seq using

699

RNA extracted from independently generated hairy roots. Raw data of RNA-Seq was

700

uploaded to NCBI (GEO accession number: GSE85077).

cufflinks

software

genes

(http://cole-trapnell-lab.github.io/cufflinks/).

using

an

online

Venn

diagram

Quantitative

Up-

or

tool reverse

701 702

Supplemental Data list:

703

Figure S1. Organ-specific expression of GmPHD6, LHP1-1 and LHP1-2.

704

Figure S2. Effector and reporter constructs used in the dual-luciferase reporter assay

705

system.

706

Figure S3. Performance of LHP1-transgenic hairy roots in response to stress.

707

Figure S4. Nucleotide sequence alignment of LHP1-1 and LHP1-2 regions coding for

708

the protein C-terminal end.

709

Figure S5. Analysis of RNA-sequencing data and quantitative RT-PCR.

710

Figure S6. Expression of ABA signaling related genes and CCDs.

711

Table S1. Statistics of clean reads in RNA sequencing.

712

Table S2. Annotation of downstream genes.

713

Table S3. Common genes were defined as genes that were up-regulated in G6-OE

714

and down-regulated in G6-RNAi.

715

Table S4. Primers used in this study.

716 31 Downloaded from on September 11, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.

717

Figure legends

718

Figure 1. Performance of GmPHD6-transgenic hairy roots and the aerial part of

719

soybean plants in response to stress.

720

(A) Performance of transgenic hairy roots after salt treatment. Soybean seedlings with

721

transgenic hairy roots were treated with 100 mM NaCl for three days. Hairy roots in

722

water

723

GmPHD6-overexpression (G6-OE) and GmPHD6 RNA interference (G6-RNAi) were

724

shown. (B) Expression Level of GmPHD6 gene in transgenic hairy roots. A soybean

725

Tubulin fragment was used as an internal control. Bars indicate SD (n=3). (C)

726

Relative growth rate of transgenic hairy roots after salt stress, Relative growth rate (%)

727

= (root elongation in salt) / (root elongation in water). Bars indicate SE (n=30).

728

Asterisks indicate significant difference (**P