Running Head: Multiple functions of Ghd7 in rice · INTRODUCTION Rice (Oryza sativa L.) is a main...

Running Head: Multiple functions of Ghd7 in rice 1

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Corresponding Author: 3

Qifa Zhang 4

National Key Laboratory of Crop Genetic Improvement and National Centre of Plant 5

Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China 6

E-mail: [email protected] 7

Phone: 86-27-8728-2429 8

Fax: 86-27-8728-7092 9

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Research Area: Genes, Development and Evolution 11

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Plant Physiology Preview. Published on January 3, 2014, as DOI:10.1104/pp.113.231308

Copyright 2014 by the American Society of Plant Biologists

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Title of article: 13

Ghd7 is a central regulator for growth, development, adaptation and responses to 14

biotic and abiotic stresses 15

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All authors’ full names: 17

Xiaoyu Weng, Lei Wang, Jia Wang, Yong Hu, Hao Du, Caiguo Xu, Yongzhong Xing, 18

Xianghua Li, Jinghua Xiao and Qifa Zhang* 19

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Affiliations: 21

National Key Laboratory of Crop Genetic Improvement and National Center of Plant 22

Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China 23

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One Sentence Summary: 25

Ghd7 regulates a range of functions in growth and development in response to 26

environmental cues to maximize the reproductive success of the rice plant. 27

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Footnotes: 29

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Financial source: This work was supported by grants from the National 863 Project 31

(2012AA10A303), the National Natural Science Foundation (31330039) and the 111 32

Project (B07041) of China, and the Bill & Melinda Gates Foundation. 33

*Corresponding author: [email protected] 34

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ABSTRACT 36

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Ghd7 has been regarded as an important regulator of heading date and yield potential 38

in rice. In the study reported in this paper, we investigated new functions of Ghd7 in 39

rice growth, development and environmental response. As a long-day dependent 40

negative regulator of heading date, the degree of phenotypic effect of Ghd7 on 41

heading date and yield traits is quantitatively related to the transcript level, and was 42

also influenced by both environmental conditions and genetic backgrounds. Ghd7 43

regulates yield traits through modulating panicle branching independent of heading 44

date. Ghd7 also regulates plasticity of tiller branching by mediating the PHYB-OsTB1 45

pathway as adaption to shade signal. The expression of Ghd7 was strongly repressed 46

by drought, ABA, JA and high temperature stress while enhanced by low temperature. 47

Over-expression of Ghd7 increased drought sensitivity while knock-down of Ghd7 48

enhanced drought tolerance. Analysis of expression profiles using gene chip revealed 49

that Ghd7 was involved in regulation of multiple processes, including flowering time, 50

hormone metabolism, biotic and abiotic stresses. This study suggested that Ghd7 51

functions to integrate the dynamic environmental inputs with phase transition, 52

architecture regulation and stress response to maximize the reproductive success of 53

the rice plant. 54

55



INTRODUCTION 56

Rice (Oryza sativa L.) is a main staple food crop feeding almost half of the world 57

population. Flowering time is one of the most important agronomic traits that 58

determines rice yield. Ghd7 encoding a CCT-domain protein is considered to be a key 59

regulator of rice specific flowering pathway and also contributes to rice yield potential 60

(Xue et al., 2008). Ghd7 controls the critical day length response of Ehd1 and florigen 61

expression through circadian gating and phytochrome action (Itoh et al., 2010; Osugi 62

et al., 2011). Two orthologs of ELF3 genes, which mediate the circadian and 63

photoperiodic regulation, act as negative regulators of Ghd7 (Zhao et al., 2012; Yang 64

et al., 2013). RID1 acts as a master switch for the transition from vegetative to 65

reproductive phase and regulates the expression of Ghd7 independent of the 66

photoperiod (Wu et al., 2008). Ehd3, which contains two plant homeodomain (PHD) 67

finger motifs and is possibly involved in the chromatin state modulation, negatively 68

regulates the transcription of Ghd7 (Matsubara et al., 2011). Lately, Hd16, a flowering 69

time QTL gene, was isolated as encoding a casein kinase I protein, that mediates the 70

phosphorylation of GHD7 and enhances the photoperiod response (Hori et al., 2013). 71

Although the complex regulation network of Ghd7 at transcription and 72

post-transcription level in flowering time control has been extensively investigated, 73

the regulation domain of Ghd7 in rice growth, development and environmental 74

response has not been adequately investigated. 75

Recent studies suggested that traditional flowering time genes may have roles in 76

plant development and stress response. In rice, two key flowering time genes, Hd1 77

and Ehd1, also control panicle development (Endo-Higashi and Izawa, 2011). In 78

Arabidopsis, the flowering promoting gene GI and the florigen genes FT and TSF 79

play a central role in drought escape response (Riboni et al., 2013). FT and TSF also 80

play a key role to link the floral transition and lateral shoot development (Hiraoka et 81

al., 2013). Molecular evidence revealed that FT and TSF proteins directly interact 82

with BRC1 protein, a homolog of TB1 (Takeda et al., 2003; Choi et al., 2012), and 83

modulates florigen activity in the axillary buds to prevent premature floral transition 84

of the axillary meristems (Niwa et al., 2013). These findings suggest that the 85



regulation of the transition to flowering also plays an important role in the modulation 86

of plant architecture plasticity and environment adaptation. 87

In this paper, we showed that the flowering time gene Ghd7 also regulates plant 88

architecture and such regulation is dependent on both genetic background and 89

environmental signaling. Ghd7 responds to various environment signals in addition to 90

day-length to regulate growth, development, biotic and abiotic stress responses. Our 91

results suggested that Ghd7 may function as a sensor for the plant to be adapted to the 92

dynamic environmental inputs and is involved in the plant architecture regulation and 93

stress response pathways. 94

95

RESULTS 96

The phenotype effect of Ghd7 is correlated with its expression level 97

Ghd7 showed pleiotropic effects on heading date, plant height and yield traits; its 98

expression was regulated by light signal and photoperiod (Xue et al., 2008; Itoh et al., 99

2010). Previously, we developed a pair of near-isogenic lines (NILs), designated 100

NIL(zs7) and NIL(mh7), with almost all the genetic background of Zhenshan 97 101

except the introgressed segment which contained the Ghd7 (Xue et al., 2008). 102

Comparison of the phenotypes of NIL(zs7), NIL(mh7) and their hybrid NIL(het) 103

showed that Ghd7 has a partial-dominant effect on flowering time, plant height and 104

yield traits (Figs. 1A, D, Table 1), consistent with previous results (Xue et al., 2008). 105

The expression level of Ghd7 in NIL(mh7) is nearly twice as that in the heterozygous 106

plants, especially at dawn (Fig. 1G). We examined the relation between the 107

expression level of Ghd7 and the phenotype in transgenic plants, in which the coding 108

sequence of Ghd7 from Minghui 63 driven by the ubiquitin promoter was transformed 109

into Hejiang 19 (HJ19) that has a non-functional allele of Ghd7 (Xue et al., 2008). Of 110

the 42 T0 plants, 37 were transgene-positive (OX-Ghd7HJ19) and exhibited the 111

expected phenotype, tall with late heading and large panicles (Supplemental Fig. S1). 112

Analysis of two random T1 families (OX-14 and OX-25) from the T0 plants showed 113

perfect co-segregation between the transgene and the phenotype (Table 1). Notably, 114

the amount of the Ghd7 transcripts was closely related to the degree of heading delay 115



and yield traits at T1 generations (Figs. 1B, 1E, 1H). These results indicated that the 116

phenotypic effect of Ghd7 is quantitatively related to the abundance of its transcript 117

and the enhanced transcript level of Ghd7 caused delayed flowering, increased plant 118

height and yield traits. 119

120

The pleiotropic effects of Ghd7 on the traits vary with genetic backgrounds and 121

environmental conditions 122

It was previously reported that enhancement of Ghd7 expression had no effect on 123

plant height and yield traits in the ehd3 mutant (Matsubara et al., 2011), and the 124

authors supposed that the function of Ghd7 also depended on other cues, such as 125

genetic background or environmental conditions. We performed transformation 126

experiments using Zhonghua 11 (ZH11), a variety with a weak-function allele of 127

Ghd7 (Xue et al., 2008). We introduced Ghd7 over-expression (OX-Ghd7ZH11) and 128

artificial miRNA (Ami-Ghd7) constructs respectively into ZH11. Seventeen of the 23 129

independent OX-Ghd7ZH11 T0 transformants showed delayed heading, and conversely 130

13 of the 21 independent Ami-Ghd7 T0 transformants showed accelerated flowering 131

(Supplemental Figs. S2A, S2B). Analysis of T1 families of OX-Ghd7ZH11 and 132

Ami-Ghd7 transformants showed perfect co-segregation between the transgene and 133

the heading date phenotype (Table 1). However, no significant increase of plant height 134

or number of spikelets per panicle was detected in the OX-Ghd7ZH11 plants 135

(Supplemental Fig. S2C, Table 1) (Seeds were sown at May 1th in Wuhan field 136

conditions, see explanation below), while Ami-Ghd7 plants showed large reduction of 137

all three traits (Supplemental Fig. S2C, Table 1). Comparison of these results with that 138

obtained from the transformants of HJ19 suggested that the pleiotropic effects of 139

Ghd7 are dependent on the genetic backgrounds, which is similar to the previous 140

finding (Xue et al., 2008). 141

The phenotypic effects of Ghd7 also varied with the environmental conditions. 142

When grown in Hainan Island in the winter nursery (natural short-day), the 143

OX-Ghd7ZH11 transgenic plants showed significant increase in plant height and 144

panicle size as well as delayed heading, compared to the wild type (Supplemental Fig. 145



S3, Supplemental Table S1). We subsequently evaluated the extent to which the 146

environments may influence the Ghd7 effects on phenotype, by examining T2 families 147

of single-copy transgenic plants of OX-Ghd7ZH11 and Ami-Ghd7 in three plantings in 148

the summer rice growing season in Wuhan. The first planting sown on April 15th and 149

second planting on May 20th subjected the plants to natural long-day conditions, while 150

the third planting sown on June 22th exposed the plants to natural short-day conditions. 151

Compared with the wild type plants, Ami-Ghd7 plants in general significantly 152

accelerated heading with decreased panicle branch number and plant height in all 153

three plantings (Table 2). The phenotype effect of Ami-Ghd7 plants was much larger 154

in the June 22th planting than the other two plantings (Figs. 1C, 1F, 1I, Table 2). 155

Conversely OX-Ghd7ZH11 plants showed delayed heading in all three planting 156

conditions (Table 2). However, the effect of the transgene on plant height and spikelet 157

number of the June 22th planting was much more drastic than the other two plantings 158

(Figs. 1C, 1F, Table 2). It should be noted that the increases of the panicle size and 159

plant height of the June 22th planting were not proportional to the length of delayed 160

heading compared with the other two plantings (Table 2). It should also be noted that 161

although no significant change was observed in the number of spikelets per panicle 162

between the OX-Ghd7ZH11 and the control in the May 20th planting, the panicle 163

architecture was changed showing an increase in the primary branch number in the 164

transgenic plant (Supplemental Table S2). These results suggested that the pleiotropic 165

effects of Ghd7 on the phenotype are influenced by the environments, and Ghd7 166

might regulate yield traits through modulating panicle architecture independent of 167

heading date. 168

169

Ghd7 regulated branching in a density dependent manner 170

Ghd7 increased the panicle branch with reduced tiller number in NIL(mh7) 171

compared to NIL(zs7) under normal field conditions (Xue et al 2008). However, we 172

observed that over-expressing Ghd7 in HJ19 increased vegetative branching in pots 173

(Supplemental Fig. S4). We supposed that the enlarged plant size of NIL(mh7) 174

relative to NIL(zs7) brings more competitive pressure which may promotes the 175



shadow avoidance signals. To test such a hypothesis, we planted NIL(mh7) and 176

NIL(zs7) under different density conditions, and found that NIL(mh7) plants had 177

significantly more tillers than NIL(zs7) plants at low density conditions (Fig. 2), 178

demonstrating that Ghd7 regulates tiller number in a density dependent manner. 179

Interestingly, there was also a significantly increase in secondary branches of the 180

panicles in NIL(mh7) relative to NIL(zs7) at low density leading to increased grain 181

number without compromising the number of primary branches (Supplemental Table 182

S3). These results suggested that Ghd7 regulates the plasticity of branch development 183

of the plant to adapt to the neighborhood environments. 184

OsTB1 was previously shown to act as a negative regulator of lateral branching in 185

rice (Takeda et al., 2003; Choi et al., 2012). We found that OsTB1 was repressed in 186

the shoot tip region in NIL(mh7) compared with NIL(zs7) (Fig. 3C). Thus we 187

generated the dsRNAi lines with reduced expression of OsTB1 (OsTB1RNAi) in the 188

ZH11 background, which showed more tillers but less panicle branching compared to 189

the control plants, in agreement with previous results (Supplemental Fig. S5) (Takeda 190

et al., 2003; Choi et al., 2012). Then we crossed the OsTB1RNAi plants to Ami-Ghd7 191

plants (with reduced tiller number and panicle branching relative to the control plants), 192

and examined the branch phenotype of the resulting F1. No significant difference in 193

tiller number of the Ami-Ghd7/OsTB1RNAi plants was detected compared to 194

OsTB1RNAi plants (Figs. 3A, 3D, 3F), whereas the flowering time of 195

Ami-Ghd7/OsTB1RNAi plants was similar to Ami-Ghd7 plants (Fig. 3D, 3E). Using 196

qRT-PCR analysis, we found a moderate increase of the OsTB1 transcript level in 197

Ami-Ghd7 plants (Fig. 3B). These results suggested that Ghd7 acts upstream of 198

OsTB1 in regulating branching. 199

200

Ghd7 mediates the PHYB-OsTB1 pathway 201

Previous studies revealed that plants’ response to shadow signals and control of 202

branching mainly depended on the PHYB-TB1 pathway (Kebrom et al., 2006; 203

Gonzalez-Grandio et al., 2013). Recently, the role of phytochrome in photoperiodic 204

flowering in rice has been elucidated (Osugi et al., 2011). The mRNA levels of both 205



Ghd7 and Ehd1 increased in the phyB mutant relative to the wild type (Osugi et al., 206

2011). To understand the effect of PHYB on the Ghd7 pathway in flowering time and 207

branch development control, we analyzed a phyB mutant in the ZH11 background. 208

The phyB mutant accelerated heading date as described before (Takano et al., 2005) 209

accompanied by reduction in tiller number in Wuhan field conditions (Supplemental 210

Fig. S6). However, we found no significant difference of Ghd7 gene expression 211

between the phyB mutant and wild type plants (Fig. 4B). We prepared an 212

anti-GHD7-specific antibody to compare the GHD7 protein level (Supplemental Fig. 213

S7). In wild type plants, the GHD7 level started to accumulate in the morning, peaked 214

at noon, gradually decreased in the afternoon till midnight and reached at a very low 215

level before the dawn (Fig. 4C). The level of GHD7 was low in the phyB mutant 216

throughout the day under long-day (Fig. 4C). This result suggested that PHYB 217

maintains the protein level of GHD7. 218

To understand the genetic interaction between PHYB and Ghd7, we generated a 219

phyB/OX-Ghd7 double mutant and compared the phenotypes of the resulting F2 220

generation. The phyB/OX-Ghd7 double mutant showed heading date similar to 221

OX-Ghd7 (Figs. 4A, 4D). Over-expression of Ghd7 partially rescued the tiller number 222

of phyB mutant (Figs. 4A, 4E). These analyses suggested that Ghd7 works 223

downstream of the PHYB. 224

225

GHD7 represses the transcriptional activity 226

It was previously reported that the middle region of the CCT domain proteins has 227

transcriptional activation activity (Tiwari et al., 2010; Wu et al., 2013). We thus 228

performed a transcriptional activation assay using the GAL4 DNA binding domain 229

and VP16 activation domain using a transient assay system with luciferase (LUC) as a 230

reporter (Fig. 5A) (Jing et al., 2013). As shown in Figure 5B, BD-GHD7 did not 231

activate the transcription of the LUC reporter gene, suggesting that GHD7 has no 232

transactivation activity in the plant cell. High LUC signal was detected in the 233

transformants of BD-VP16 construct, due to the transcriptional activation by the 234

VP16 domain (Fig. 5B). However, the activity of LUC was drastically reduced by 235



GHD7 (BD-VP16-GHD7) (Fig. 5B). These results suggested that GHD7 has intrinsic 236

transcriptional repression activity in vivo. 237

238

Expression of Ghd7 is regulated by environmental signals 239

An analysis of Ghd7 spatial expression profile revealed that the expression of 240

Ghd7 was mainly detected in the emerged leaf blade, whereas it was virtually absent 241

in other tissues assayed even in the pre-emerged immature leaf blade surrounded by 242

leaf sheath (Supplemental Fig. S8A). In emerged leaf blade, Ghd7 transcripts 243

displayed a gradient with much higher transcript accumulation in the leaf tip than the 244

leaf base (Supplemental Fig. S8B). The Ghd7 transcript levels were relatively 245

constant at vegetative, reproductive and ripening stages in the leaf blade (Fig. 6A), 246

which was similar to Hd1 (Supplemental Fig. S9A). However, the expression of Ehd1 247

and Hd3a was low during the first 7 weeks, but increased at reproductive and ripening 248

stages (Fig. 6B, Supplemental Fig. S9B). 249

We analyzed the DNA sequence of the promoter region of Ghd7 and found a 250

number of cis-elements including ones involved in stress response such as ABRE 251

element, CBF element and hormone response elements like MYB/MYC recognition 252

site and ABA/JA response elements (Fig. 6C) (Finkelstein and Lynch, 2000; Abe et al., 253

2003; Brown et al., 2003; Simpson et al., 2003; Svensson et al., 2006). Thus we 254

assayed the Ghd7 expression in rice seedling treated with different phytohormones 255

and drought stress. The accumulation of Ghd7 mRNA was induced by cold treatments, 256

but repressed by drought, ABA, JA and high temperature treatments (Fig. 6D). The 257

expression of Ghd7 was slightly affected by ACC and SA treatments (Fig. 6D). These 258

results suggested that Ghd7 was involved in response to various environmental 259

signals in addition to photoperiod. 260

261

Ghd7 regulates the transcriptomes of multiple processes 262

To gain clues to downstream genes regulated by Ghd7, we performed a 263

microarray analysis using Affimetrix rice gene chips. Young leaves (35 days after 264

germination) and developing panicles (0.1 cm) from field-grown OX-Ghd7HJ19 265



transgenic and wild-type plants were used to isolate RNA for chip analysis. With a 266

threshold of 2-fold change, a total of 256 and 622 genes were up- and down-regulated, 267

respectively, in the leaves of OX-Ghd7HJ19 plants (Figs. 7A, 7D, Supplemental Table 268

S4). In the young panicles of OX-Ghd7HJ19 plants, 177 genes were up-regulated and 269

303 down-regulated compared with wild type (Figs. 7B, 7E, Supplemental Table S5). 270

These analyses supported the previous conclusion that Ghd7 mainly plays an 271

inhibitory role in gene expression. 272

Expression of several flowering-related genes was altered in OX-Ghd7HJ19 plants 273

both in young leaves and developing panicles. Ehd1 and FT-like genes were 274

down-regulated in leaves of OX-Ghd7HJ19 plants, consistent with previous results 275

(Supplemental Table S4) (Xue et al., 2008; Itoh et al., 2010). Expression of a large 276

number of MADS-box genes appeared to be altered in both leaves and panicles in 277

OX-Ghd7HJ19 plants, mostly down-regulated, including OsMADS1, OsMADS14, 278

OsMADS18 and OsMADS34 in leaves, which regulate reproductive transition and 279

panicle architecture (Supplemental Table S4) (Lee et al., 2004; Kobayashi et al., 280

2012). However, OsMADS55, which was considered as a negative regulator of 281

flowering associated with ambient temperature, was significantly up-regulated both in 282

leaves and panicles in OX-Ghd7HJ19 plants (Supplemental Table S4) (Lee et al., 2012). 283

Expression of many genes involved in hormone metabolism and signaling 284

pathways was affected in OX-Ghd7HJ19 plants. Expression of an auxin-inducible gene 285

Oshox1, which regulates the sensitivity of polar auxin transport (Scarpella et al., 286

2002), increased in OX-Ghd7HJ19 plants (Fig. 7C). The cytokinin oxidase gene 287

OsCKX2, which negatively regulates rice grain number (Ashikari et al., 2005), was 288

down-regulated in OX-Ghd7HJ19 plants (Fig. 7C). Ethylene and gibberellin (GA) 289

contribute to internode elongation (Iwamoto et al., 2011). The transcript abundance of 290

OsACO1, a key enzyme gene involved in ethylene synthesis pathway (Iwamoto et al., 291

2010), was up-regulated in OX-Ghd7HJ19 plants (Fig. 7C). While the gibberellin 292

2-oxidase gene OsGA2ox6, which controls plant height and tiller number (Lo et al., 293

2008; Huang et al., 2010), was repressed in OX-Ghd7HJ19 plants (Fig. 7C). 294

Consistently, OsCKX2 and OsACO1 were also down-regulated and up-regulated in 295



the Ami-Ghd7 plants respectively (Fig. 7C). These results suggested that Ghd7 was 296

involved in regulating multiple hormonal pathways. 297

Many transcription factor (TF) families also appeared to be affected in 298

OX-Ghd7HJ19 plants, most notably the AP2, HLH, MYB, WRKY, and Zinc finger TFs 299

both in leaves and panicles (Supplemental Table S4, S5). Some TF families are tissue 300

specifically down-regulated in OX-Ghd7HJ19 plants, such as CCT-domain genes in 301

leaves and TCP and YABBY genes in panicles (Supplemental Table S4, S5). The CCT 302

genes were implicated in flowering time control by photoperiod and circadian 303

pathways (Valverde, 2011). The YABBY and TCP genes were shown to participate in 304

the activities controlling lateral organs as well as the shoot apical meristem (SAM) 305

(Dai et al., 2007; Martin-Trillo and Cubas, 2010). These results suggested that Ghd7 306

played a different role in vegetative and reproductive organs by regulating various 307

transcription networks. 308

309

Ghd7 is involved in stress-response pathways and ROS homeostasis 310

Interestingly, we found that many Ghd7-regulated genes are involved in pathways 311

of responses to abiotic and biotic stresses. Among them, OsDREB1A and OsPR4, 312

which play a role in cold and drought stress respectively (Dubouzet et al., 2003; Wang 313

et al., 2011), were both significantly up-regulated in OX-Ghd7HJ19 plants (Fig. 7F). 314

OsDREB1A was down-regulated in Ami-Ghd7 plants, but not OsPR4 (Fig. 7F). We 315

applied drought stress to OX-Ghd7HJ19 and Ami-Ghd7 plants, and found that 316

Ami-Ghd7 plants showed enhanced drought tolerance, while OX-Ghd7HJ19 plants 317

were more sensitive to drought (Fig. 8A, 8B). The results indicated that Ghd7 was 318

indeed involved in regulation of drought stress response. 319

Reactive oxygen species (ROS) serve as important signaling molecules that 320

participate in response to both biotic and abiotic stresses (Sagi et al., 2004; Gechev et 321

al., 2006; Miller et al., 2008). OsMT2b is a ROS scavenger and functions as the signal 322

in resistance response (Wong et al., 2004). Our analysis showed that OsMT2b was 323

up-regulated in OX-Ghd7HJ19 plants, but down-regulated in Ami-Ghd7 plants (Fig. 324

7F). OsrbohE and RACK1A genes, which are involved in ROS production during the 325



immune response, were down-regulated in OX-Ghd7HJ19 plants (Fig. 7F) (Yoshiaki et 326

al., 2005; Nakashima et al., 2008), while both of them were up-regulated in 327

Ami-Ghd7 plants (Fig. 7F). Finally, a group of ROS homeostasis-related genes and 328

wall-associated kinase (WAK) family genes showed at least a 2-fold change in 329

expression in OX-Ghd7HJ19 plants (Supplemental Table S6, S7). These data suggested 330

that Ghd7 affects the expression of genes whose proteins might be components in the 331

network of ROS homeostasis and responds to biotic stresses by changing the cell wall 332

components. 333

334

DISCUSSION 335

Unlike animals, plants have a remarkable ability to alter their development in 336

response to myriad exogenous and endogenous signals in the life cycle. Previously, 337

we cloned the QTL gene Ghd7, which acts as an important regulator of heading date 338

and yield potential in rice (Xue et al., 2008). More recent works showed that Ghd7 339

mainly functions as a flowering repressor under long-day conditions and was 340

regulated by light- and circadian clock-dependent gating (Xue et al., 2008; Itoh et al., 341

2010; Osugi et al., 2011). Besides the light signal, another important environmental 342

aspect, temperature, regulated Ghd7 expression as well (Song et al., 2012). 343

In this study, we found that the Ghd7 transcript was regulated by various 344

environmental signals such as light, temperature, abiotic and biotic stresses, and 345

subsequently the expression level of Ghd7 regulated the growth and development of 346

the rice plant. ABA is a regulatory molecule involved in drought stress tolerance and 347

JA is involved in plant’s response to biotic stresses (Yamaguchi-Shinozaki and 348

Shinozaki, 2006; Robert-Seilaniantz et al., 2007). We showed that ABA, JA and 349

drought treatments strongly repressed the Ghd7 expression, which may be related to 350

the response of the plant to quickly end the life cycle in adverse conditions in order to 351

escape or avoid stresses. Moreover, the results that Ghd7 regulated the stress-related 352

genes and ROS homeostasis genes suggested that Ghd7 might be involved in these 353

stress pathways as well. 354

Recently, Matsubara et al., (2011) reported that a PHD-finger gene Ehd3 355



repressed the Ghd7 transcription. However, they observed no substantial increase in 356

seed productivity in the ehd3 mutant, despite increased Ghd7 expression (Matsubara 357

et al., 2011). In this study we found that over-expression of Ghd7 in ZH11 delayed the 358

heading date regardless of the planting conditions, but drastically increased the yield 359

traits in June but not April or May plantings under natural field conditions in Wuhan. 360

The results implied that a certain combination of environmental conditions may be 361

required for Ghd7 to increase the yield traits of the rice plant. Thus Ghd7 might not 362

only function as a flowering time regulator, but also as a sensor of the environmental 363

signals for the plant to dynamically regulate growth, development, morphology, 364

architecture and stress responses (Fig. 9). 365

Tiller and panicle branches are lateral organs at vegetative and reproductive 366

stages in rice, respectively. Panicle branching is often associated with the flowering 367

time, likely because of longer vegetative periods. Studies have also revealed that some 368

flowering time genes, such as Hd1 and Ehd1, control panicle development in rice, 369

independently of flowering time control (Endo-Higashi and Izawa, 2011). Meanwhile, 370

several genes, such as Gn1a, SP1 and DEP1 exclusively alter the number of panicle 371

branches, without simultaneous changes of flowering time or the tiller number in rice 372

(Ashikari et al., 2005; Huang et al., 2009; Li et al., 2009). Tiller branching is 373

modulated by both genetic factor and environmental conditions. The mutations of 374

MOC1, LAX1 and LAX2 lead to a reduced number of both tillers and panicle branches 375

(Komatsu et al., 2003; Li et al., 2003; Tabuchi et al., 2011). While in the case of d and 376

OsTB1 mutants, the effect of the genes on tillers and panicle branches is opposite to 377

each other; an increase in the tiller number is accompanied by a decrease in the 378

panicle branches (Takeda et al., 2003; Lin et al., 2009; Choi et al., 2012). 379

Recently, it was shown that two florigen genes, FT and TSF, modulate lateral 380

shoot outgrowth in Arabidopsis (Hiraoka et al., 2013). Moreover, these two florigen 381

proteins interact with BRC1, which was considered as Arabidopsis TB1 clade gene, to 382

repress the floral transition of the axillary buds in Arabidopsis (Niwa et al., 2013). 383

These results suggested a potential link between flowering time control and branching 384

development. We previously showed that Ghd7 increased panicle branching but 385



decreased tiller branching. The present result suggested that Ghd7 positively regulates 386

both tiller and panicle branches in a density dependent manner, suggesting that Ghd7 387

is involved in regulating the plasticity of branch development for adaptation to 388

different environmental conditions. This process is partly regulated by PHYB by 389

maintaining the GHD7 protein. Then Ghd7 repressed OsTB1 expression, partly 390

through the GA signaling (Lo et al., 2008) and enhanced the expression of 391

OsMADS57, which is considered as an interaction protein of OsTB1 (Guo et al., 2013), 392

to control tiller branching. In panicle branch, many genes involved in specifying 393

meristem and lateral organ identity, including TCP genes, SPL genes and YABBY 394

genes, were regulated by Ghd7. Thus the effects of Ghd7 on multiple traits can be 395

explained by delaying the phase transition and increasing the lateral organ growth 396

activity. We propose that Ghd7 played a key role to integrate the floral transition and 397

lateral branch development in response to environmental cues to maximize the 398

reproductive success of the rice plant (Fig. 9). 399

400

401

MATERIALS AND METHODS 402

Growth conditions of the rice plants 403

The rice plants examined under natural field conditions were grown in Wuhan 404

(Huazhong Agricultural University, E 114°21', N 30°28') and Hainan Island (Lingshui 405

County, E 110°01', N 18°30'), China. The rice growing season in Wuhan summer has 406

in general relative high temperature and long-day conditions (unless otherwise 407

specified), while the winter nursery in Hainan has relatively low temperature and 408

short-day conditions. Germinated seeds were sown in the seed beds (late April to late 409

June in Wuhan, and Middle to late November in Hainan) and seedlings of one month 410

old were transplanted to the fields. The planting density was normally 16.5 cm 411

between plants in a row, and the rows were 26 cm apart. For the density experiment, 412

this normally density was regarded as the high density conditions and in the low 413

density conditions the plants were 70 cm apart in a row, and the distance between 414

rows were 30 cm. Field management, including irrigation, fertilizer application and 415



pest control, followed essentially the normal agricultural practice. 416

417

Phenotypic data collection 418

Heading date was the day when the first panicle of the plant emerged. Total 419

number of spikelets on the main panicle of the plant was counted about 10 days after 420

heading. Plant height was measured from the ground to the tip of the tallest tiller of 421

the plant. 422

423

Generation of Constructs and Transformation 424

To construct the OX-Ghd7HJ19 vector, the open reading frame of Ghd7 was 425

amplified by PCR using primers OX-F and OX-R (Supplemental Table 8), containing 426

restriction sites for KpnI and BamHI respectively for subcloning. cDNA was cloned 427

into the pCAMBIA1301U vector and then transformed into HJ19. 428

To construct the OX-Ghd7ZH11 vector, the Ghd7 promoter region was amplified 429

with PRO-F and PRO-R primer containing KpnI and BamHI sites respectively and 430

subcloned into the pCAMBIA1301 vector (Supplemental Table 8). Then the 431

full-length cDNA of Ghd7 was amplified by PCR using primers ORF-F and ORF-R 432

containing BamHI and HindIII sites respectively (Supplemental Table 8) and inserted 433

into the pCAMBIA1301 vector to fuse with the promoter region to generate the 434

OX-Ghd7ZH11 construct and then transformed into ZH11. 435

To construct the Ami-Ghd7 vector, we used a customized version of the original 436

Web MicroRNA Designer platform (WMD) to design amiRNA sequences (21 mers) 437

based on the TIGR5 rice genome annotation. We selected the most suitable amiRNA 438

candidates suggested by WMD that have good hybridization properties to the target 439

mRNAs with single target in the rice genome, with no off-target effect to other genes. 440

The primary amiRNA construct was amplified with Ami-Ghd7-I, Ami-Ghd7-II, 441

Ami-Ghd7-III and Ami-Ghd7-IV primers (Supplemental Table 8) which was 442

engineered from pNW55 as previously described (Warthmann et al., 2008). The 443

fusion product of 554 bp was cloned into pGEM-T Vector (Promega), excised with 444

KpnI and BamHI and cloned into the pCAMBIA1301U vector and then transformed 445



into ZH11. 446

To construct the OsTB1RNAi vector, a 484-bp fragment of OsTB1 was amplified 447

by PCR using primers OsTB1RNAi-F and OsTB1RNAi-R (Supplemental Table 8). 448

OsTB1RNAi-F primer contained SpeI and KpnI sites and OsTB1RNAi-R primer 449

contained SacI and BamHI sites, for subcloning into the pDS1301 vector that was a 450

modified version of pCAMBIA1301 (Yuan et al., 2007). 451

All the constructs were independently introduced into the Agrobacterium strain 452

EHA105, and transformation was done as described previously (Ge et al., 2006). 453

454

RNA Extraction and qRT-PCR 455

We isolated total RNA using an RNA extraction kit (TRIzol reagent; Invitrogen) 456

according to the manufacturer’s instructions. For quantitative real-time PCR, 457

approximately 3 μg total RNA was reverse-transcribed using SuperScript II reverse 458

transcriptase (Invitrogen) in a volume of 100 μL to obtain cDNA. We carried out 459

quantitative real-time PCR in a total volume of 25 μL containing 2 μL of the 460

reverse-transcribed product above, 0.25 mM gene-specific primers, and 12.5 μL 461

SYBR Green Master Mix (Applied Biosystems) on an Applied Biosystems 7500 462

Real-Time PCR System according to the manufacturer’s instructions. Primer pairs for 463

qRT–PCR analysis are listed in Supplemental Table 8. The measurements were 464

obtained using the relative quantification method (Livak and Schmittgen, 2001). 465

466

Purification of Recombinant Protein 467

To construct the recombinant MBP-GHD7 vector, the open reading frame of 468

Ghd7 was amplified with MBP-GHD7-F and MBP-GHD7-R primers containing 469

EcoRI and BamHI sites and subcloned into the pMAL vector. MBP and MBP-GHD7 470

recombinant fusion proteins were induced by isopropyl b-D-1-thiogalactopyranoside 471

and expressed in the Escherichia coli BL21 (DE3) strain. The proteins were then 472

purified by MBP beads following the manufacturer’s instructions. 473

474

Antibody Production and immunoblotting 475



The peptide corresponding to amino acids 243 to 257 of GHD7 476

(CTYVDPSRLELGQWFR) conjugated with KLH was synthesized, and polyclonal 477

antibody was raised in rabbit. Rice leaf total protein extraction was performed as 478

described (Li et al., 2011). Proteins were boiled in SDS loading buffer, separated by 479

10% SDS-PAGE gels, and blotted onto polyvinylidene fluoride membranes. The 480

proteins were then incubated with anti-Ghd7 (1:200 dilution) or anti-HSP (1:5000 481

dilution) (Li et al., 2011) and subsequently the horseradish peroxidase–conjugated 482

goat-anti-rabbit secondary antibody (Abcam) according to the manufacturer’s 483

instructions. The protein bands were visualized by the standard ECL kit (Thermo 484

Scientific Pierce) and the signal was exposed with X-ray film. 485

486

LUC Activity Assay 487

To determine the transcriptional activation activity of GHD7, the full-length 488

GHD7 fused with the GAL4 DNA binding domain (BD-GHD7) was co-transformed 489

into Arabidopsis protoplasts with a reporter construct containing the 4× UAS region 490

and mini 35S promoter sequence fused to LUC cDNA. To analyze the transcriptional 491

repression activity of GHD7, the full-length GHD7 was fused with the GAL4-VP16 492

domain (BD-VP16-GHD7), which is a widely used transcriptional activator, and 493

co-transformed into Arabidopsis protoplasts with the reporter construct. The LUC 494

activity assay was performed as previously reported (Tang et al., 2012). LUC reporter 495

activity was detected with a luminescence kit using the LUC assay substrate 496

(Promega). Relative reporter gene expression levels are expressed as the ratio of LUC 497

to GUS. 498

499

Stress Treatments of Plant Materials 500

To check the expression level of the Ghd7 under various abiotic stresses or 501

phytohormone treatments, rice plants of NIL(mh7) were grown in hydroponic culture 502

medium for about 3 weeks in a phytotron (14L/10D and 32°C/26°C). Seedlings at the 503

four-leaf stage were treated with abiotic stresses, including drought (removing the 504

water supply under phytotron conditions, 14L/10D and 32°C/26°C), cold (seedlings 505



were transferred to a phytotron at 14L/10D and 10°C/10°C) and heat (seedlings were 506

transferred to a phytotron at 14L/10D and 42°C/42°C). For phytohormone treatments, 507

20 μM ABA, 0.5 mM jasmonic acid, 0.1 mM SA, 0.1 mM ACC was individually 508

added to the culture medium. The sample was collected at the designated time points 509

(0 min, 30 min, 6 h and 12 h). 510

To test the drought stress tolerance of transgenic plants at the seedling stage, 511

transgenic positive and wild-type plants (30 plants each, three repeats) were grown in 512

a half-and-half manner in barrels filled with sandy soil. Drought stress testing was 513

conducted at the four-leaf stage, following the procedure as described previously 514

(Tang et al., 2012). 515

516

Microarray Analysis 517

RNA samples used for microarray analysis were prepared from young leaves in 518

vegetative stage (35-days old) and developing panicles (0.1 cm in length) of 519

OX-Ghd7HJ19 transgenic and wild-type plants grown under normal field conditions 520

with two biological replicates. RNA isolation, purification and Affymetrix microarray 521

hybridization were carried out using the protocol of the Affymetrix GeneChip service 522

(CapitalBio). The microarray analysis was conducted according to the previously 523

described process (Yang et al., 2012), and the data can be found in the 524

GenBank/EMBL data libraries under series accession numbers GSE51616. 525

526

SUPPLEMENTAL DATA 527

Supplemental Figure S1. T0 generation plants of OX-Ghd7HJ19 planted under natural 528

long-day field conditions in Wuhan. 529

Supplemental Figure S2. T0 and T1 generation plants of OX-Ghd7ZH11 and 530

Ami-Ghd7 planted under natural long-day field conditions in Wuhan. 531

Supplemental Figure S3. T2 generation plants of OX-Ghd7ZH11 and Ami-Ghd7 532

planted under natural short-day field conditions in Hainan Island. 533

Supplemental Figure S4. OX-Ghd7HJ19 plants that showed increase in tiller number 534

at vegetative stage. 535



Supplemental Figure S5. Phenotype of OsTB1RNAi plants in ZH11 background 536

planted under natural long-day field conditions in Wuhan. 537

Supplemental Figure S6. phyB mutants in ZH11 background planted under natural 538

long-day field conditions in Wuhan. 539

Supplemental Figure S7. Detection of GHD7-MBP protein by GHD7 antibody. 540

Supplemental Figure S8. Expression feature of Ghd7. 541

Supplemental Figure S9. Expression patterns of Hd1 and Hd3a at various 542

developmental stages. 543

Supplemental Table S1. Performance of OX-Ghd7ZH11 and Ami-Ghd7 plants under 544

natural short-day conditions in Hainan Island. 545

Supplemental Table S2. The panicle architecture of OX-Ghd7ZH11 and Ami-Ghd7 546

plants with different sowing times in Wuhan field conditions. 547

Supplemental Table S3. Branch number in panicles of NIL(zs7) and NIL(mh7) under 548

low and high planting densities. 549

Supplemental Table S4. The differentially regulated genes in young leaves revealed 550

by microarray analysis. 551

Supplemental Table S5. The differentially regulated genes in developing panicles 552

revealed by microarray analysis. 553

Supplemental Table S6. ROS homeostasis-related genes revealed by microarray 554

analysis of young leaves. 555

Supplemental Table S7. WAK family genes revealed by microarray analysis of 556

young leaves. 557

Supplemental Table S8. The primers used in this work. 558

559

ACKNOWLEDGMENTS 560

We acknowledge Professor Hongwei Xue for providing the phyB mutant seeds in 561

ZH11 background. We are grateful to Dr. Lin Shao and Dr. Yidan Ouyang for their 562

critical comments on the manuscript. 563

564



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759

760

FIGURE LEGENDS 761

Figure 1. Phenotypes and Ghd7 expression levels of the various genotypes generated 762

in this work. 763

(A) and (D) The whole plants (A) and main panicles (D) of NIL(zs7), NIL(het) and 764

NIL(mh7) under natural long-day conditions in Wuhan taken at maturity. 765

(B) and (E) The whole plants (B) and main panicles (E) of Ghd7-overexpressor in 766

HJ19 background under natural long-day conditions in Wuhan. 767

(C) and (F) The whole plants (C) and main panicles (F) of OX-Ghd7 and Ami-Ghd7 768

in ZH11 background sown in June in Wuhan. Bars = 50cm in (A) to (C) and bars = 769

10cm in (D) to (F). 770

(G) Diurnal expression analysis of Ghd7 in leaf blades of the NILs. The samples were 771

collected at 40 days after germination (DAG) under natural long-day conditions in 772

Wuhan and used for RNA preparation. The numbers below the x axis indicate 773

zeitgeber times (ZTs) of the day. The white bar indicates the light period, and the 774



black bar indicates the dark period. Each points and error bars indicate average values 775

and SE, respectively, based on three biological repeats. 776

(H) and (I) Expression levels of Ghd7 in transgenic plants in HJ19 background (H) 777

and ZH11 background (I). Leaf blades from plants of 30 DAG were collected at 2 h 778

after dawn and used for RNA preparation. Bars and error bars indicate average values 779

and SE, respectively, based on three biological repeats. 780

781

Figure 2. The effect of Ghd7 on tillering in different density conditions. 782

(A) Tillers of NIL(zs7) and NIL(mh7) under low and high density conditions under 783

natural long-day conditions in Wuhan. Bars = 10cm. 784

(B) Tiller number of NIL(zs7) and NIL(mh7) at 68 DAG (the heading date of 785

NIL(zs7)) and 91 DAG (the heading date of NIL(mh7)) (three different plantings with 786

30 plants each). Error bars indicate SE. 787

788

Figure 3. Genetic interaction between Ghd7 and the OsTB1 pathway. 789

(A) and (D) Plants of wild type ZH11 (a), Ami-Ghd7 (b), Ami-Ghd7/OsTB1RNAi (c) 790

and OsTB1RNAi (d) at vegetative stage (A) and reproductive stage (D) under natural 791

long-day conditions in Wuhan. Bar = 20cm in (A) and bar = 40cm in (D). 792

(B) Expression levels of OsTB1 in wild type (a), Ami-Ghd7 (b), 793

Ami-Ghd7/OsTB1RNAi (c) and OsTB1RNAi (d) plants. The samples were collected 794

at 30 DAG under natural long-day conditions in Wuhan and used for RNA preparation. 795

Bars and error bars indicate average values and SE, respectively, based on three 796

biological repeats. 797

(C) Expression levels of OsTB1 in NIL(zs7) and NIL(mh7). The samples were 798

collected at 35 DAG under natural long-day conditions in Wuhan and used for RNA 799

preparation. Bars and error bars indicate average values and SE, respectively, based 800

on three biological repeats. 801

(E) and (F) The number of days to heading (E) and the number of tillers (F) of wild 802

type (a), Ami-Ghd7 (b), Ami-Ghd7/OsTB1RNAi (c) and OsTB1RNAi (d) plants 803

under long-day conditions in Wuhan (n ≥ 15 each). Error bars indicate SE. 804



805

Figure 4. Genetic interaction of Ghd7 and the PHYB pathway. 806

(A) Plants of wild type ZH11 (PHYBPHYB), phyB mutant (phyBphyB), 807

phyBphyB/OX-Ghd7, phyBPHYB/OX-Ghd7 and OX-Ghd7 (PHYBPHYB/OX-Ghd7) 808

at reproductive stage under natural long-day conditions in Wuhan. Bar = 40cm. 809

(B) Diurnal expression analysis of Ghd7 in leaf blades of the phyB mutant and ZH11. 810

The samples for RNA preparation were collected at 35 DAG under natural long-day 811

conditions in Wuhan. The numbers below the x axis indicate ZTs of the day. The 812

white bar indicates the light period, and the black bar indicates the dark period. Each 813

points and error bars indicate average values and SE, respectively, based on three 814


(C) Protein levels of GHD7 in leaf blades of the phyB mutants and ZH11. The 816

samples for protein preparation were collected at the same conditions as in (B). GHD7 817

protein was detected using anti-GHD7 antibody. Heat shock protein (HSP) antibody 818

was used as the reference for western blotting. Two independent experiments 819

produced consistent results. 820

(D) and (E) The heading date (D) and tiller number (E) of wild type ZH11 821

(PHYBPHYB), phyB mutant (phyBphyB), phyBphyB/OX-Ghd7, 822

phyBPHYB/OX-Ghd7 and OX-Ghd7 (PHYBPHYB/OX-Ghd7) plants under natural 823

long-day conditions in Wuhan (n ≥ 10 each). Error bars indicate SE. 824

825

Figure 5. Repression activity of GHD7. 826

(A) Diagram of various constructs used in this assay. 827

(B) Relative luciferase activities in Arabidopsis protoplasts after transfection with 828

reporter plasmids and effectors of various constructs. Bars and error bars indicate 829

average values and SE, respectively, based on three biological repeats. 830

831

Figure 6. Expression of Ghd7 in the lifecycle and in response to various environment 832

signals. 833

(A) and (C) Expression patterns of Ghd7 and Ehd1 at various developmental stages. 834



The leaf blades of the plants were collected for RNA preparation at every ten days 835

under long-day conditions in Wuhan. The numbers below the x axis indicate the DAG. 836



(B) Putative stress related cis-elements in the promoter region of Ghd7. 839

(D) Expression level of Ghd7 under stress and phytohormone treatments including 840

drought, ABA, JA, cold, heat, ACC and SA (0, 0.5, 6 and 12 h). Bars and error bars 841

indicate average values and SE, respectively, based on three biological repeats. 842

843

Figure 7. The profiles of genes regulated by Ghd7. 844

(A) and (B) Scatterplots of expression profiles of the complete gene set in leaf (A) 845

and panicle (B) in OX-Ghd7HJ19 compared with the wild type (WT). The x and y axes 846

indicate the chip hybridization signal in the overexpressor and the wild type, 847

respectively. The pink and green dots indicate the probe sets with OX:wild type signal 848

ratios of greater than 2 or less than 0.5, respectively. 849

(C) The differential regulation patterns of some hormone related genes in 850

OX-Ghd7HJ19 (up) and Ami-Ghd7 (down) plants. Bars and error bars indicate average 851

values and SE, respectively, based on three biological repeats. 852

(D) and (E) Expression patterns of all the differentially regulated genes in leaf (D) and 853

panicle (E) in OX-Ghd7HJ19 plants relative to the wild type. 854

(F) The differential regulation patterns of abiotic and biotic stress responsive genes in 855

OX-Ghd7HJ19 (up) and Ami-Ghd7 (down) plants. Bars and error bars indicate average 856

values and SE, respectively, based on three biological repeats. 857

858

Figure 8. Response of Ghd7 to drought stress. 859

(A) Phenotypes of OX-Ghd7HJ19 and Ami-Ghd7 under drought stress. Bars = 10cm. 860

(B) Survival rate of OX-Ghd7HJ19 and Ami-Ghd7 after drought stress (n = 30 each). 861



864



Figure 9. A schematic illustration of the Ghd7 functions learned from this study. 865

Ghd7 functions to link the dynamic environmental inputs with phase transition, 866

architecture regulation and stress response to maximize the reproductive success of 867

the rice plant. 868

869



Table 1 870

Performance of NILs and T1 family of transgene positive and negative plants in HJ19 871

and ZH11 backgrounds in Wuhan under natural long-day field conditions. 872

Data presented in the first three rows are from the three NILs planted in randomized 873

complete block design with three replications. A, B and C indicate ranking by Duncan 874

test at P < 0.01 (starting from A, B is significantly different from A, and C is 875

significantly different from B). (+) and (–) indicate transgene-positive and 876

transgene-negative, respectively. ** Statistically significant at P < 0.01 by t-test. 877

878

879

880

881

882

883

884

No.

plants Plant

height (cm) No. days to

heading No. spikelets

on the main panicle

NIL(zs7) 20 93.2 (A) 66.3 (A) 125.6 (A)

NIL(het) 20 114.0 (B) 80.1 (B) 194.7 (B)

NIL(mh7) 20 127.9 (C) 87.8 (C) 230.1 (C)

T1 generation

HJ19 10 59.3±1.1 52.4±1.4 56.4±6.9

OX-Ghd7HJ19 (-) 10 58.7±1.0 50.2±1.4 57.9±8.2

OX-Ghd7HJ19-14 (+) 20 78.5±2.0** 82.1±2.4** 112.4±10.2**

OX-Ghd7HJ19-25 (+) 20 99.1±3.1** 103.2±3.1** 157.1±16.3**

T1 generation

ZH11 16 103.7±2.0 70.8±1.5 167.7±10.8

OX-Ghd7ZH11 (+) 16 105.6±2.3 87.8±2.8** 176.9±15.0**

OX-Ghd7ZH11 (-) 12 104.6±1.2 68.8±1.9 160.6±10.0

Ami-Ghd7 (+) 16 79.4±3.1** 57.1±1.7** 92.3±8.8**

Ami-Ghd7 (-) 11 104.8±1.3 67.9±1.9 163.6±8.4



Table 2 885

Performance of the single copy transgenic plants of T2 generation of OX-Ghd7ZH11 886

and Ami-Ghd7 with different sowing times in Wuhan field conditions. 887

Sowing times

No. plants

Plant height (cm)

No. days to heading

No. spikelets on the main panicle

WT April 15th

20 106.8±1.5 79.7±1.8 210.6±13.4

OX-Ghd7ZH11 20 102.5±2.7** 97.1±1.5** 236.4±15.7**

Ami-Ghd7 20 92.5±2.3** 71.7±1.7** 167.6±16.9**

WT

May 20th

20 95.0±1.9 69.3±1.7 174.1±11.2

OX-Ghd7ZH11 20 102.5±4.1** 86.2±1.9** 176.4±25.3

Ami-Ghd7 20 74.3±2.6** 56.6±1.2** 104.7±15.7**

WT

June 22th

20 89.3±2.8 63.1±2.0 166.5±14.0

OX-Ghd7ZH11 20 112.5±3.9** 85.9±2.4** 267.5±21.7**

Ami-Ghd7 20 68.7±2.9** 47.8±1.6** 62.5±8.1**

** Statistically significant at P < 0.01 by t-test. 888



NIL(zs7) NIL(het) NIL(mh7) HJ19 OX-14 OX-25 ZH11 OX-Ghd7ZH11 Ami-Ghd7

NIL(zs7) NIL(het) NIL(mh7) HJ19 OX-14 OX-25 ZH11 OX-Ghd7ZH11 Ami-Ghd7

A B C

D E F

Bar=50cm Bar=50cm Bar=50cm

Bar=10cm Bar=10cm Bar=10cm

0

100

200

300

400

HJ19 OX-14 OX-250.0

0.5

1.0

1.5

150

200

250

ZH11 OX-Ghd7ZH11 Ami-Ghd7

Rel

ativ

e ex

pres

sion

I

Figure 1. Phenotypes and Ghd7 expression levels of the various genotypes generated in this work.(A) and (D) The whole plants (A) and main panicles (D) of NIL(zs7), NIL(het) and NIL(mh7) under natural long-day conditions in Wuhan taken at maturity.(B) and (E) The whole plants (B) and main panicles (E) of Ghd7-overexpressor in HJ19 background under natural long-day conditions in Wuhan.(C) and (F) The whole plants (C) and main panicles (F) of OX-Ghd7 and Ami-Ghd7 in ZH11 background sown in June in Wuhan. Bars = 50cm in (A) to (C) and bars = 10cm in (D) to (F).(G) Diurnal expression analysis of Ghd7 in leaf blades of the NILs. The samples were collected at 40 days after germination (DAG) under natural long-day conditions in Wuhan and used for RNA preparation. The numbers below the x axis indicate zeitgeber times (ZTs) of the day. The white bar indicates the light period, and the black bar indicates the dark period. Each points and error bars indicate average values and SE, respectively, based on three biological repeats.(H) and (I) Expression levels of Ghd7 in transgenic plants in HJ19 background (H) and ZH11 background (I). Leaf blades from plants of 30 DAG were collected at 2 h after dawn and used for RNA preparation. Bars and error bars indicate average values and SE, respectively, based on three biological repeats.

ZT2 ZT6 ZT10 ZT14 ZT18 ZT22

0

0.5

1

1.5

2

2.5

3

8:30 12:30 16:30 20:30 0:30 4:30

NIL(zs7)

NIL(het)

NIL(mh7)

Rel

ativ

e ex

pres

sion

Rel

ativ

e ex

pres

sion

G H



NIL(zs7)

NIL(mh7)

High density Low densityN

umbe

r of t

illers

0

10

20

30

40

High density (68 DAG)

High density (91 DAG)

Low density (68 DAG)

Low density (91 DAG)

NIL(zs7) NIL(mh7)

Figure 2. The effect of Ghd7 on tillering in different density conditions. (A) Tillers of NIL(zs7) and NIL(mh7) under low and high density conditions under natural long-day conditions in Wuhan. Bars = 10cm.(B) Tiller number of NIL(zs7) and NIL(mh7) at 68 DAG (the heading date of NIL(zs7)) and 91 DAG (the heading date of NIL(mh7)) (three different plantings with 30 plants each). Error bars indicate SE.

A

BBar=10cm



a b c d

a b c d

0

0.4

0.8

1.2

0

0.4

0.8

1.2

1.6

a b c d

0

10

20

30

40

a b c d50

60

70

80

a b c d

Rel

ativ

e ex

pres

sion

Rel

ativ

e ex

pres

sion

Num

ber o

f tille

rs

Num

ber o

f day

s

to h

eadi

ng

Figure 3. Genetic interaction between Ghd7 and the OsTB1 pathway.(A) and (D) Plants of wild type ZH11 (a), Ami-Ghd7 (b), Ami-Ghd7/OsTB1RNAi (c) and OsTB1RNAi (d) at vegetative stage (A) and reproductive stage (D) under natural long-day conditions in Wuhan. Bar = 20cm in (A) and bar = 40cm in (D).(B) Expression levels of OsTB1 in wild type (a), Ami-Ghd7 (b), Ami-Ghd7/OsTB1RNAi (c) and OsTB1RNAi (d) plants. The samples were collected at 30 DAG under natural long-day conditions in Wuhan and used for RNA preparation. Bars and error bars indicate average values and SE, respectively, based on three biological repeats.(C) Expression levels of OsTB1 in NIL(zs7) and NIL(mh7). The samples were collected at 35 DAG under natural long-day conditions in Wuhan and used for RNA preparation. Bars and error bars indicate average values and SE, respectively, based on three biological repeats.(E) and (F) The number of days to heading (E) and the number of tillers (F) of wild type (a), Ami-Ghd7 (b), Ami-Ghd7/OsTB1RNAi (c) and OsTB1RNAi (d) plants under long-day conditions in Wuhan (n ≥ 15 each). Error bars indicate SE.

A B C

D E FNIL(zs7) NIL(mh7)

Bar=20cm

Bar=40cm



Rel

ativ

e ex

pres

sion

GHD7 (ZH11)

HSP (ZH11)

GHD7 (phyB)

HSP (phyB)

Num

ber o

f tille

rs

Hea

ding

dat

e (D

ays)

PHYBPHYB phyBphyB phyBphyB phyBPHYB PHYBPHYB(ZH11) (phyB) /OX-Ghd7 /OX-Ghd7 /OX-Ghd7

0123456789

ZT1 ZT4 ZT7 ZT10 ZT13 ZT16 ZT19 ZT22

phyB

ZH11

ZT1 ZT4 ZT7 ZT10 ZT13 ZT16 ZT19 ZT22

50

60

70

80

90

PHYBPHYB(ZH11)

phyBphyB(phyB)

phyBphyB/OX-Ghd7

phyBPHYB/OX-Ghd7

PHYBPHYB/OX-Ghd7

0

2

4

6

8

10

12

PHYBPHYB(ZH11)

phyBphyB(phyB)

phyBphyB/OX-Ghd7

phyBPHYB/OX-Ghd7

PHYBPHYB/OX-Ghd7

A

B C

D E

Figure 4. Genetic interaction of Ghd7 and the PHYB pathway.(A) Plants of wild type ZH11 (PHYBPHYB), phyB mutant (phyBphyB), phyBphyB/OX-Ghd7, phyBPHYB/OX-Ghd7 and OX-Ghd7 (PHYBPHYB/OX-Ghd7) at reproductive stage under natural long-day conditions in Wuhan. Bar = 40cm. (B) Diurnal expression analysis of Ghd7 in leaf blades of the phyB mutant and ZH11. The samples for RNA preparation were collected at 35 DAG under natural long-day conditions in Wuhan. The numbers below the x axis indicate ZTs of the day. The white bar indicates the light period, and the black bar indicates the dark period. Each points and error bars indicate average values and SE, respectively, based on three biological repeats.(C) Protein levels of GHD7 in leaf blades of the phyB mutants and ZH11. The samples for protein preparation were collected at the same conditions as in (B). GHD7 protein was detected using anti-GHD7 antibody. Heat shock protein (HSP) antibody was used as the reference for western blotting. Two independent experiments produced consistent results.(D) and (E) The heading date (D) and tiller number (E) of wild type ZH11 (PHYBPHYB), phyB mutant (phyBphyB), phyBphyB/OX-Ghd7, phyBPHYB/OX-Ghd7 and OX-Ghd7 (PHYBPHYB/OX-Ghd7) plants under natural long-day conditions in Wuhan (n ≥ 10 each). Error bars indicate SE.

Bar=40cm



0 10 20 30

GAL4VP16 Ghd7

GAL4VP16

GAL4DB Ghd7

GAL4DB

Effector for Transcription repressor

Effector for Transactivation Activity

Reporter

4 X UAS Mini 35S Luciferase

35S TEV GAL4DB Ghd7

Poly A signal

Poly A signal

35S TEV GAL4VP16 Ghd7 Poly A signal

A

B

Figure 5. Repression activity of GHD7. (A) Diagram of various constructs used in this assay.(B) Relative luciferase activities in Arabidopsis protoplasts after transfection with reporter plasmids and effectors of various constructs. Bars and error bars indicate average values and SE, respectively, based on three biological repeats.

Relative activity (LUC/GUS)



0

0.1

0.2

0.3

0.4

20 30 40 50 60 70 80 90 100

0

1

2

3

20 30 40 50 60 70 80 90 100

Rel

ativ

e ex

pres

sion

Rel

ativ

e ex

pres

sion

Rel

ativ

e ex

pres

sion

DAG

DAG

Ghd7

Ehd1

A B

CD

Figure 6. Expression of Ghd7 in the lifecycle and in response to various environment signals.(A) and (C) Expression patterns of Ghd7 and Ehd1 at various developmental stages. The leaf blades of the plants were collected for RNA preparation at every ten days under long-day conditions in Wuhan. The numbers below the x axis indicate the DAGs. Bars and error bars indicate average values and SE, respectively, based on three biological repeats.(B) Putative stress related cis-elements analysis in the promoter region of Ghd7.(D) Expression level of Ghd7 under stress and phytohormone treatments including drought, ABA, JA, cold, heat, ACC and SA (0, 0.5, 6 and 12 h). Bars and error bars indicate average values and SE, respectively, based on three biological repeats.

0

0.5

1

1.5

2

2.5

3

CK DR ABA JA Cold Heat ACC SA

0min 30min6hrs 12hrs



Rel

ativ

e ex

pres

sion

Leaf

256

622

WT

OX

-Ghd

7HJ1

9

177

303

Panicle

WT WTOX-Ghd7HJ19 OX-Ghd7HJ19

WT

Rel

ativ

e ex

pres

sion

Rel

ativ

e ex

pres

sion

Rel

ativ

e ex

pres

sion

OX

-Ghd

7HJ1

9

Leaf Panicle

0

1

2

3

4

5

6

OsHox1 OsCKX2 OsACO1 OsGA2ox6

HJ19 OX-Ghd7HJ19

01234567

OsHox1 OsCKX2 OsACO1 OsGA2ox6

ZH11 Ami-Ghd7

0

2

4

6

8

OsDREB1A OsMT2b OsPR4 OsrbohE RACK1A

HJ19 OX-Ghd7HJ19

0

1

2

3

4

5

OsDREB1A OsMT2b OsPR4 OsrbohE RACK1A

ZH11 Ami-Ghd7

A B C

D E

F

Figure 7. The profiles of genes regulated by Ghd7. (A) and (B) Scatterplots of expression profiles of the complete gene set in leaf (A) and panicle (B) in OX-Ghd7HJ19 compared with the wild type (WT). The x and y axes indicate the chip hybridization signal in the overexpressor and the wild type, respectively. The pink and green dots indicate the probe sets with OX:wild type signal ratios of greater than 2 or less than 0.5, respectively.(C) The differential regulation patterns of some hormone related genes in OX-Ghd7HJ19 (up) and Ami-Ghd7 (down) plants. Bars and error bars indicate average values and SE, respectively, based on three biological repeats.(D) and (E) Expression patterns of all the differentially regulated genes in leaf (D) and panicle (E) in OX-Ghd7HJ19 plants relative to the wild type. (F) The differential regulation patterns of abiotic and biotic stress responsive genes in OX-Ghd7HJ19 (up) and Ami-Ghd7 (down) plants. Bars and error bars indicate average values and SE, respectively, based on three biological repeats.

rep 1 rep 2 rep 1 rep 2 rep 1 rep 2 rep 1 rep 2



Before drought stress After recovery

HJ19 OX-Ghd7HJ19

ZH11 Ami-Ghd7

HJ19 OX-Ghd7HJ19

ZH11 Ami-Ghd7

Sur

viva

l rat

e af

ter

Dro

ught

stre

ss (%

) S

urvi

val r

ate

afte

rD

roug

ht s

tress

(%)

Figure 8. Response of Ghd7 to drought stress.(A) Phenotypes of OX-Ghd7HJ19 and Ami-Ghd7 under drought stress. Bars = 10cm.(B) Survival rate of OX-Ghd7HJ19 and Ami-Ghd7 after drought stress (n = 30 each). Bars and error bars indicate average values and SE, respectively, based on three biological repeats.

A BBar=10cmBar=10cm

Bar=10cmBar=10cm

0

20

40

60

80

100

0

20

40

60

80

100

ZH11 Ami-Ghd7

HJ19 OX-Ghd7HJ19



Environmental cues Day length, temperature, shade signal

drought stress and hormones (ABA, JA)

GHD7

Ghd7

Phase transition

Architecture regulation

Stress responses

PHYs Others?

Figure 9. A schematic illustration of the Ghd7 functions learned from this study.Ghd7 functions to link the dynamic environmental inputs with phase transition, architecture regulation and stress response to maximize the reproductive success of the rice plant.



Running Head: Multiple functions of Ghd7 in rice · INTRODUCTION Rice (Oryza sativa L.) is a main...

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Transcript of Running Head: Multiple functions of Ghd7 in rice · INTRODUCTION Rice (Oryza sativa L.) is a main...