1

RESEARCH ARTICLE 1

The Arabidopsis Nodulin Homeobox Factor AtNDX Interacts with 2

AtRING1A/B and Negatively Regulates Abscisic Acid Signaling 3

Yujuan Zhu1, 2#

, Xiaoying Hu1,,# Ying Duan

1, 3#, Shaofang Li

1, Yu Wang

1, Amin Ur 4

Rehman1, Junna He

1, Jing Zhang

1, Deping Hua

1, Li Yang

1, Li Wang

1, Zhizhong Chen

1, 5

Chuanyou Li4, Baoshan Wang

5, Chun-Peng Song

6, Qianwen Sun

7, Shuhua Yang

1, 6

Zhizhong Gong1, *

7

1State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, 8 China Agricultural University, Beijing, 100193, China 9

2Lingnan Guangdong Laboratory of Modern Agriculture, Genome Analysis Laboratory of the 10 Ministry of Agriculture, Agricultural Genomics Institute at Shenzhen, Chinese Academy of 11 Agricultural Sciences, Shenzhen, 518100, China 12

3 Key Laboratory of Biology and Genetic Improvement of Horticultural Crops of the Ministry 13 of Agriculture, Institute of Vegetables and Flowers, Chinese Academy of Agricultural 14 Sciences, Beijing, 100081, China 15

4State Key Laboratory of Plant Genomics, National Centre for Plant Gene Research, Institute 16 of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, 17 China 18

5Key Lab of Plant Stress Research, College of Life Science, Shandong Normal University, 19 Ji’nan 250000, China 20

6Collaborative Innovation Center of Crop Stress Biology, Henan Province, Institute of Plant 21 Stress Biology, Henan University, Kaifeng 475001, China 22

7Center for Plant Biology and Tsinghua-Peking Joint Center for Life Sciences, School of Life 23 Sciences, Tsinghua University, Beijing 100084, China 24

#: These authors contributed equally to this work. 25

*Corresponding author: gongzz@cau.edu.cn 26

27

Short title: AtNDX and PRC1 core components AtRING1A/B in ABA signaling 28

One-sentence summary: Arabidopsis nodulin homeobox protein AtNDX interacts with the 29 Polycomb Repressive Complex 1 (PRC1) core components AtRING1A and AtRING1B and 30 negatively regulates ABI4 expression during ABA signaling. 31

The author responsible for distribution of materials integral to the findings presented in this 32 article in accordance with the policy described in the Instructions for Authors 33 (www.plantcell.org) is Zhizhong Gong, gongzz@cau.edu.cn. 34

35

ABSTRACT 36

The phytohormone abscisic acid (ABA) and the Polycomb group (PcG) proteins have 37

key roles in regulating plant growth and development; however, their interplay and 38

underlying mechanisms are not fully understood. Here, we identified an Arabidopsis 39

thaliana nodulin homeobox (AtNDX) protein as a negative regulator in the ABA 40

signaling pathway. AtNDX mutants are hypersensitive to ABA, as measured by 41

Plant Cell Advance Publication. Published on January 9, 2020, doi:10.1105/tpc.19.00604

©2020 American Society of Plant Biologists. All Rights Reserved

2

inhibition of seed germination and root growth, and the expression of AtNDX is 42

downregulated by ABA. AtNDX interacts with the Polycomb Repressive Complex 1 43

(PRC1) core components AtRING1A and AtRING1B in vitro and in vivo, and 44

together, they negatively regulate the expression levels of some ABA-responsive 45

genes. We identified ABA-INSENSITIVE (ABI4) as a direct target of AtNDX. AtNDX 46

directly binds the downstream region of ABI4 and deleting this region increases the 47

ABA sensitivity of primary root growth. Furthermore, ABI4 mutations rescue the 48

ABA-hypersensitive phenotypes of ndx mutants and ABI4-overexpressing plants are 49

hypersensitive to ABA in primary root growth. Thus, our work reveals the critical 50

functions of AtNDX and PRC1 in some ABA-mediated processes and their regulation 51

of ABI4. 52

53

INTRODUCTION 54

The phytohormone abscisic acid (ABA) regulates a variety of developmental 55

processes including seed dormancy, seed germination, seedling growth, flowering and 56

plant senescence (Cutler et al., 2010; Zhu, 2016; Nonogaki, 2019). ABA also plays 57

crucial and generally protective roles in various abiotic stress responses including 58

drought, high salt, and low temperature (Cutler et al., 2010; Zhu, 2016; Qi et al., 59

2018). In the ABA signaling pathway, after perceiving ABA, ABA receptors 60

PYR/PYLs/RCARs interact with and inhibit the negative regulators of the clade 61

A-type protein phosphatases 2C (PP2Cs), which releases the inhibitory effect of these62

PP2Cs on downstream protein kinases such as SnRK2 (SUCROSE 63

NONFERMENTING1-RELATED PROTEIN KINASES2).2/2.3/2.6 (OST1, OPEN 64

STOMATA1), GUARD CELL HYDROGEN PEROXIDE-RESISTANT1 (GHR1) 65

and calcium-dependent protein kinases (CPKs) (Ma et al., 2009; Park et al., 2009; 66

Cutler et al., 2010; Zhu, 2016) (Hua et al., 2012; Qi et al., 2018). Some protein 67

kinases such as SnRKs then phosphorylate and activate downstream targets such as 68

the SLOW ANION CHANNEL-ASSOCIATED 1 (SLAC1), which regulates stomatal 69

movement, or transcription factors such as the basic leucine zipper (bZIP)-family 70

proteins ABFs (ABRE binding factors)/AREBs (including ABF1, ABF2/AREB1, 71

ABF3 and ABF4/AREB2) (Choi et al., 2000; Uno et al., 2000) and ABI5 (ABA 72

INSENSITIVE 5) (Finkelstein and Lynch, 2000), which regulate the expression of 73

ABA-responsive genes. 74

3

In addition to bZIP transcription factors, many other transcription factors such as 75

MYB, MYC, NAC (NAM, ATAF1/2 and CUC2), WRKY (The 76

tryptophan-arginine-lysine-tyrosine), ZFHD1 (ZINC-FINGER HOMEODOMAIN), 77

NF-Y (NUCLEAR FACTOR-Y) and ARF2 (AUXIN RESPONSE FACTOR 2) are 78

involved in the ABA signaling pathway (Ren et al., 2010; Fujita et al., 2011; Wang et 79

al., 2011; Rushton et al., 2012; Singh and Laxmi, 2015; Promchuea et al., 2017). 80

Early studies of ABA-insensitive (ABI) mutants in seed germination identified several 81

genes such as ABI1, ABI2, ABI3, ABI4, and ABI5. ABI1 and ABI2, which encode 82

PP2Cs, are key negative regulators in the early ABA signaling pathway. ABI3 83

encodes a transcription factor with a B3 domain that shares high sequence similarity 84

with maize (Zea mays) Viviparous1 (Giraudat et al., 1992). ABI4 encodes an 85

APETALA2 (AP2)-type transcription factor (Finkelstein et al., 1998), which can bind 86

to target genes by recognizing CE1-like cis-elements (CACCG and CCAC motif) and 87

act as either an activator or a repressor depending on the context (Kerchev et al., 2011; 88

Shu et al., 2013; Wind et al., 2013; Huang et al., 2017). ABI4 expression is hormone 89

regulated, and its protein stability is affected by ABA and gibberellic acid (GA) in 90

opposite ways (Shkolnik-Inbar and Bar-Zvi, 2010; Shu et al., 2016). Several 91

transcription factors have been reported to directly regulate ABI4 expression, such as 92

ABI4 itself and WRKY8, which positively regulate ABI4 expression (Bossi et al., 93

2009; Chen et al., 2013), and RAV1 (Related to ABI3/VP1) (Feng et al., 2014) and 94

BASIC PENTACYSTEINE (BPC) family proteins, which negatively regulate ABI4 95

(Mu et al., 2017). BPCs can bind the ABI4 promoter and recruit Polycomb Repressive 96

Complex 2 (PRC2), thus repressing ABI4 expression through the histone H3 lysine 27 97

trimethylation (H3K27me3) epigenetic modification (Mu et al., 2017). 98

Polycomb group proteins (PcGs) are the major epigenetic machinery executing 99

transcriptional repression and developmental regulation in animals and plants 100

(Calonje, 2014; Mozgova and Hennig, 2015; Zhou et al., 2018). The two best- 101

characterized PcG complexes to date are PRC1 and PRC2 (Mozgova and Hennig, 102

2015). PRC1 deposits histone H2A monoubiquitination (H2Aub) and mediates 103

chromatin compaction of its target genes (Calonje, 2014; Wang and Shen, 2018), and 104

PRC2 catalyzes H3K27me3 (Mozgova and Hennig, 2015). PRC1 components were 105

initially identified in Drosophila melanogaster by genetic approaches. The canonical 106

Drosophila PRC1 consists of Polycomb (Pc), Polyhomeotic (Ph), Posterior sex combs 107

4

(Psc), and dRing1, also known as Sex combs extra (Sce). These have multiple 108

homologs in mammals, resulting in different possible combinations of PcGs (Shao et 109

al., 1999; Francis et al., 2001). Arabidopsis thaliana has no Ph protein, and the LIKE 110

HETEROCHROMATIN PROTEIN 1 (LHP1), a plant homolog of animal HP1, 111

functions like Pc in recognizing and colocalizing with H3K27me3 genome-wide 112

(Turck et al., 2007; Zhang et al., 2007). EARLY BOLTING IN SHORT DAYS (EBS) 113

and SHORT LIFE (SHL) interact with EMBRYONIC FLOWER 1 (EMF1), a 114

functional equivalent of the Psc C-terminal region (Psc-CTR), to form a bivalent 115

chromatin reader complex for both H3K27me3 and H3K4me3 that mediate switching 116

between repressed and active chromatin during plant development (Li et al., 2018; 117

Yang et al., 2018). The most conserved PRC1 components in plants are the 118

RING-finger proteins RING1 and BMI1, corresponding to dRing1 and the N-terminus 119

of Psc in Drosophila, respectively (Molitor and Shen, 2013; Merini and Calonje, 120

2015). In Arabidopsis, two RING1 homologs (AtRING1A and AtRING1B) and three 121

BMI1 homologs (AtBMI1A, AtBMI1B and AtBMI1C) have been identified based on 122

a unique domain architecture that contains a conserved RING finger domain in the 123

N-terminal region and a ubiquitin-like domain named RAWUL (Ring-finger And 124

WD40 associated Ubiquitin-Like) in the C-terminal region (Sanchez-Pulido et al., 125

2008; Xu and Shen, 2008). AtRING1A and AtRING1B interact with themselves, with 126

each other, and with LHP1, EMF1, AtBMI1A, AtBMI1B, and AtBMI1C (Wang and 127

Shen, 2018). The two AtRING1A/B proteins and the three AtBMI1A/B/C proteins 128

have been shown to have E3 ligase activity in vitro and in vivo (Bratzel et al., 2010; 129

Bratzel et al., 2012). 130

Target selection by PcGs at various developmental stages is critical for the 131

functions of targets. In Arabidopsis, both cis-element and DNA binding factors are 132

required for recruiting PRC2 (Xiao et al., 2017). Besides the classical hierarchical 133

model in which PRC1 is recruited by the binding of Pc to the H3K27me3 marker 134

deposited by PRC2, numerous factors involved in the recruitment of PRC1 have 135

emerged in mammals (Blackledge et al., 2015). In plants, many proteins interact with 136

PRC1 components EMF1, LHP1, AtRING1A and AtBMI1A/B/C (Li et al., 2016; 137

Wang and Shen, 2018; Zhu et al., 2019). AtRING1A interacts with ALFIN1-like 138

PHD-domain H3K4me3-binding protein AL6 and directly regulates the expression of 139

ABI3 and DELAY OF GERMINATION 1 (DOG1 ) (Molitor et al., 2014), CLF 140

5

(CURLY LEAF, a PRC2 component catalyzing H3K27-methylation) (Xu and Shen, 141

2008), and FAS1 (Chromatin Assembly Factor-1 subunit) during DNA replication 142

(Jiang and Berger, 2017). The dissection of different PcG component regulons reflects 143

the important roles of PRC1 in repressing gene expression during seed maturation and 144

vegetative development, and suggests that DNA binding protein may play a crucial 145

role in recruiting PcG to specific targets in plants (Wang and Shen, 2018). Until now, 146

however, no transcription factor has been identified that interacts with AtRING1A/B 147

(Wang and Shen, 2018). 148

In our previous studies, we used a root-bending assay to study plant response to 149

ABA, and we identified several ABA overly-sensitive (abo) mutants that show 150

hypersensitivity to ABA with respect to inhibition of primary root growth (Liu et al., 151

2010; Ren et al., 2010; Wang et al., 2011; He et al., 2012; Yang et al., 2014; 152

Promchuea et al., 2017; Wang et al., 2018). The cloned genes include ABO3, which 153

encodes a WRKY transcription factor (Ren et al., 2010); ARF2 (Wang et al., 2011), 154

ABO5, and ABO8, which encode two pentatricopeptide repeat proteins localized in 155

mitochondria (Liu et al., 2010; Yang et al., 2014); ABO6, which encodes a 156

DEXH-box RNA helicase in mitochondria (He et al., 2012); and EAR1 (ENHANCER 157

OF ABA CO-RECEPTOR1), which encodes a novel protein that enhances the activity 158

of PP2Cs (Wang et al., 2018). 159

Here we characterized two mutant alleles of an Arabidopsis thaliana nodulin 160

homeobox homologous gene, AtNDX. NDX was previously isolated from a soybean 161

(Glycine max) nodule-specific expression library (Jorgensen et al., 1999) and 162

represents a small gene family in different plants. The Arabidopsis thaliana genome 163

has only one AtNDX gene. AtNDX can bind the single-stranded DNA at the 3′end of 164

Arabidopsis FLOWERING LOCUS C (FLC) and control expression of FLC and the 165

antisense transcript COOLAIR (Sun et al., 2013). Our results demonstrate that the 166

expression of AtNDX is downregulated by ABA and that AtNDX directly interacts 167

with AtRING1A and AtRING1B. These proteins co-regulate the expression of some 168

common ABA-responsive genes. Further, AtNDX directly binds the downstream 169

region of ABI4 and represses its expression, and mutation of ABI4 could recover the 170

ABA-hypersensitive phenotype of ndx mutants in both seed germination and primary 171

root growth. 172

173

6

RESULTS 174

AtNDX is a negative regulator of ABA-mediated inhibition of seed germination 175

and primary root growth. 176

A root-bending assay (Yin et al., 2009) was employed to screen for ABA 177

overly-sensitive mutants in an ethyl methyl sulfonate (EMS)-mutagenized 178

Arabidopsis M2 population, in which we identified two ABA overly-sensitive mutant 179

alleles, ndx-5 and ndx-6. Genetic analysis indicated that ndx-5 and ndx-6 are recessive 180

mutations in a single nuclear gene. ndx-5 and ndx-6 were backcrossed to wild-type 181

Columbia four times before performing the following analyses. 182

First, we quantified the ABA-induced inhibition of primary root growth in ndx 183

mutants and the wild type. The primary root growth was measured after 5-d-old 184

seedlings were moved from Murashige and Skoog (MS) medium to MS medium or 185

MS medium supplemented with different concentrations of ABA. The ndx mutants 186

and the wild type showed no difference in primary root growth on MS medium. 187

However, ABA-induced inhibition of root growth was significantly more severe in 188

ndx mutants than in the wild type. Here, we used ear1-1 for comparison of ABA 189

sensitivity (Figure 1A and 1B) (Wang et al., 2018). 190

Root growth integrates root cell division, differentiation, and elongation. 191

Therefore, we further determined the meristem zone (MZ) cell number and 192

differentiation zone (DZ) cell length in ndx-5 and wild type, which reflect the root cell 193

division activity and mature cell size, respectively. Consistent with the primary root 194

growth, both the MZ cell number and DZ cell length were inhibited by ABA to a 195

greater extent in the ndx mutant than in the wild type (Supplemental Figure 1A-1D). 196

Then we investigated the ABA sensitivity of the seed germination and seedling 197

establishment in ndx mutants and the wild type. With increasing concentrations of 198

ABA, the percentage of seedlings with green cotyledons in ndx mutants was reduced 199

significantly more than those of wild type, while there was no obvious difference 200

between ndx and wild type on MS medium without ABA (Figure 1C-1D, 201

Supplemental Figure 2A-2D). 202

We also detected the expression of ABA response genes, including ABI3 and 203

ABI4, and found the expression levels of these two genes were higher in ndx mutants 204

than the wild type (Supplemental Figure 2E). However, we did not find any difference 205

7

between the wild type and ndx mutants in a detached leaf water loss assay and 206

stomata number on leaves (Supplemental Figure 2F and 2G), suggesting that NDX is 207

not involved in water loss under drought stress. These results demonstrate that 208

AtNDX is a negative regulator of ABA signaling in seed germination and primary 209

root growth, but not in ABA-promoted stomatal closure. 210

Isolation of AtNDX by map-based cloning 211

We identified the mutation in ndx-5 by map-based cloning. AtNDX was delimited to 212

BAC clones T4I9 and F4C21 on chromosome IV (Supplemental Figure 3A). 213

Sequencing of putative genes in this region identified a G1911 to A1911 mutation 214

(counting from the first putative ATG of AT4G03090) in AT4G03090 (previously 215

named AtNDX, Supplemental Figure 3A) (Mukherjee et al., 2009). We compared the 216

sequences amplified from cDNAs of ndx-5 and the wild type and found that the point 217

mutation affected the acceptor splicing between the seventh intron and the eighth 218

exon and created a new acceptor site upstream of the original one, resulting in an 219

early putative stop codon in the new transcript (Supplemental Figure 3B). 220

Map-based cloning narrowed down the ndx-6 mutation to the same region as ndx-5. 221

Sequencing of AT4G03090 in ndx-6 revealed a mutation from G569 to A569, which is 222

suspected to change the donor splicing site between the third exon and third intron 223

from GT to AT. Transcript analyses indicated that cDNA from ndx-6 contained an 224

extra third intron that creates an early putative stop codon in the new transcript 225

(Supplemental Figure 3C). We also obtained a previously described AtNDX mutant, 226

enhancer of coolair1-4 (eoc1-4), here renamed ndx-4 (Sun et al., 2013). In the ndx-4 227

mutant, AtNDX is disrupted by a T-DNA insertion (Supplemental Figure 3A) (Sun et 228

al., 2013). ndx-4 showed similar ABA sensitivity to ndx-5 with respect to root growth 229

and seedling establishment (Figure 1A-1D). 230

Immunoblot assays using NDX antibodies indicated that the AtNDX level was 231

significantly reduced in the ndx-4, ndx-5 and ndx-6 mutants compared to the wild type, 232

GST-AtNDX-C from Escherichia coli was used as the positive control to evaluate the 233

specificity of NDX antibodies (Supplemental Figure 3E). Given that the phenotypes 234

of ndx-5 are weaker than those of ndx-6 for both flowering time (Supplemental Figure 235

3F) and ABA sensitivity, ndx-5 is assumed to produce a truncated protein with some 236

residual function. 237

8

An 8-kb fragment that includes the wild-type AT4G03090 gene and contains 238

about 3000 bp upstream of the first putative ATG to 1000 bp downstream of the 239

putative TGA stop codon complemented the ABA-sensitive phenotypes of ndx-5 in 240

several independent lines (two independent lines are shown in Supplemental Figure 241

4A and 4B). In addition, overexpressing AtNDX-MYC under control of a 35S promoter 242

or AtNDX-Flag driven by the native promoter also complemented the ABA-sensitive 243

root phenotype of ndx-5 or ndx-4 (Supplemental Figure 4C-4F). However, we found 244

that these overexpression lines were not more resistant to ABA than the wild type in 245

terms of primary root growth, suggesting that only a certain amount of AtNDX 246

protein is needed for its full functioning in ABA-mediated primary root growth, or the 247

protein did not accumulate much, as AtNDX is likely regulated by 26S proteasome 248

pathway as shown below. These results indicate that AT4G03090 is the AtNDX gene. 249

ABA negatively regulates the expression of AtNDX 250

To elucidate the role of AtNDX in the ABA response, we first examined the effect of 251

ABA treatment on AtNDX expression and AtNDX protein levels. Transcript analysis 252

by reverse transcription-quantitative PCR (RT-qPCR) showed that the expression of 253

AtNDX was significantly repressed by ABA (Figure 2A). We then assayed transgenic 254

plants transformed with the native AtNDX promoter (proAtNDX) driving a GUS 255

reporter gene and found that ABA treatment greatly reduced GUS expression as 256

observed by GUS staining (Figure 2B). We also observed GFP fluorescence in 257

transgenic plants carrying proAtNDX:AtNDX-GFP and found that 30 μM ABA 258

treatment significantly reduced GFP expression (Figure 2C and 2D). 259

To explore whether the AtNDX protein level is affected by ABA, we analyzed the 260

total protein extracts from seedlings with or without ABA treatment. Immunoblotting 261

analysis with anti-NDX antibodies indicated that endogenous AtNDX protein was 262

reduced under ABA treatment. When we used intracellular protein synthesis inhibitor 263

cycloheximide (CHX) or CHX plus ABA to treat the seedlings to block protein 264

biosynthesis in order to see the ABA effect on AtNDX protein, the protein reduction 265

caused by ABA was no longer detected (Figure 2E). These results indicate that ABA 266

negatively regulates AtNDX at the transcriptional level, but not at the 267

posttranscriptional level. At the same time, we found that AtNDX was accumulated 268

when seedlings were treated with the proteasome inhibitor MG132. This result 269

suggests that AtNDX is regulated by the 26S proteasome degradation pathway (Figure 270

9

2E). Here, GST-AtNDX-C was used as a positive control. 271

AtNDX interacts with PRC1 core components AtRING1A and AtRING1B 272

To further clarify the working mechanism of AtNDX, we attempted to identify the 273

proteins interacting with AtNDX through a yeast two-hybrid screening. A normalized 274

Arabidopsis yeast two-hybrid cDNA library was used as the prey, and screened with 275

binding domain (BD) fused with AtNDX as the bait. Two clones encoding AtRING1A 276

fragments and one clone encoding an AtRING1B fragment were identified among 277

about 100 sequenced clones encoding putative interactors. We then verified their 278

interaction with AtNDX using the full-length AtRING1A or AtRING1B in the yeast 279

two-hybrid assay (Figure 3A). 280

We next tested whether AtNDX and AtRING1A or AtRING1B interact in vivo by 281

using a co-immunoprecipitation (Co-IP) assay. Proteins were extracted from 282

protoplasts transiently expressing HA-Flag-AtNDX and AtRING1A- or 283

AtRING1B-GFP and used for Co-IP. The results indicated that AtRING1A-GFP and 284

AtRING1B-GFP co-immunoprecipitated HA-Flag-AtNDX (Figure 3B). Then, we 285

used GFP beads to immunoprecipitate proteins extracted from 10-d-old transgenic 286

seedlings (two lines) containing AtRING1A-GFP driven by a super promoter to detect 287

whether the endogenous AtNDX protein could coimmunoprecipitate with 288

AtRING1A-GFP. An immunoblot assay with anti-NDX antibodies showed that 289

endogenous AtNDX protein coimmunoprecipitated with AtRING1A-GFP (Figure 290

3C). 291

To examine the natural interaction between AtNDX and AtRING1A/B, we used 292

anti-NDX antibodies in immunoprecipitation experiments to identify 293

AtNDX-associated proteins from 10-d-old seedlings. Following mass spectrometry 294

analysis, we identified AtRING1A and AtBMI1A, suggesting that AtNDX is 295

associated with PRC1 core components in vivo (Figure 3D). Therefore, we tested 296

whether there were direct interactions between AtNDX and other PRC1 components 297

such as AtBMI1A, AtBMI1B, AtBMI1C, AtEMF1 or LHP1 using the yeast 298

two-hybrid assay. However, we did not find any interaction (Supplemental Figure 5). 299

To determine which parts of AtRING1A/B and AtNDX confer their physical 300

interaction, different deletions were tested in the yeast two-hybrid assay. Deletion 301

analysis revealed that the C-terminus of AtRING1A/B, containing the RAWUL 302

10

domain, is sufficient to bind AtNDX (Figure 3E), while the full-length AtNDX protein 303

is required for their interaction (Figure 3F). Together, our data demonstrate that 304

AtNDX interacts with AtRING1A/B in vitro and in vivo. 305

The atring1a atring1b double mutants are hypersensitive to ABA in primary root 306

growth and seedling establishment 307

To investigate the role of AtRING1A/B in the ABA response, we examined the 308

phenotype of atring1a and atring1b single and double mutants on MS medium 309

containing ABA. Single mutants for atring1 or atbmi1 do not show apparent 310

morphological phenotypes except for a late flowering phenotype of atring1a (Shen et 311

al., 2014). However, the atring1a/b and atbmi1a/b double mutants displayed strong 312

embryonic defects during post-germination (see Supplemental Figure 6 for mutant 313

information), which is consistent with the derepression of several key regulatory 314

genes implicated in embryogenesis and stem cell activity (Xu and Shen, 2008; Bratzel 315

et al., 2010; Chen et al., 2010). The expression of AtRING1A/B was not affected in 316

ndx mutants and the expression of AtNDX was not affected in the ring mutants 317

(Supplemental Figure 6B-6D). The atring1a and atring1b-2 single mutants displayed 318

similar ABA sensitivity to the wild type (Supplemental Figure 7A-7D and 8A-8D). 319

We then selected 5-day-old atring1a atring1b double mutants according to the 320

abnormal cotyledon phenotypes on MS medium and transferred them onto medium 321

supplemented with or without ABA for 4 days. Although atring1a atring1b mutants 322

displayed various root lengths on MS medium, their relative root growth (i.e., growth 323

on ABA medium relative to growth on ABA-free medium) was significantly more 324

sensitive to ABA than that of the wild type (Figure 4A-4C). 325

Since the atring1a atring1b double mutant is sterile, we were unable to test the 326

seed cotyledon greening phenotype on ABA-containing medium. We then used a 327

weak double mutant, ring1a-2 ring1b-3, in which a T-DNA fragment is inserted in the 328

promoter region of AtRING1A, resulting in reduced expression of AtRING1A 329

compared to the wild type and a more uniform phenotype (Supplemental Figure 6) (Li 330

et al., 2017). The atring1a-2 atring1b-3 double mutant was significantly more 331

sensitive to ABA with respect to inhibition of seedling establishment than the wild 332

type, and ear1-1 served as the ABA hypersensitive control (Figure 4D-4G). In 333

conclusion, these results revealed that AtRING1A and AtRING1B negatively regulate 334

ABA responses with respect to seedling establishment. 335

11

To examine the genetic interaction between AtNDX and AtRING1A/B, we 336

constructed ndx-5 atring1a and ndx-5 atring1b-2 double mutants and tested their 337

phenotypes on ABA medium. The ndx-5 atring1a mutant displayed slightly but not 338

significantly increased ABA sensitivity with respect to inhibition of root growth and 339

substantially increased ABA sensitivity with respect to inhibition of seedling 340

establishment (Supplemental Figure 7A-7D), while the ndx-5 atring1b-2 double 341

mutant showed similar sensitivity to the ndx-5 single mutant (Supplemental Figure 342

8A-8D). The ndx-5 atring1a double mutant even exhibited shorter roots and smaller 343

cotyledons on MS medium compared with wild type (Supplemental Figure 7C). The 344

additive effects of AtNDX and AtRING1A could be due to each protein having 345

specific additional targets. Our data suggest that AtRING1A plays a broader, more 346

important role than AtRING1B as demonstrated by its role in flowering control (Shen 347

et al., 2014). Collectively, the above results indicate that the AtNDX and 348

AtRING1A/B proteins are involved in some ABA-mediated responses. 349

AtNDX and AtRING1A/B co-regulate the expression of ABI4 and some 350

ABA-responsive genes 351

Because AtNDX and AtRING1A/B interact with each other in vivo and both mutants 352

are hypersensitive to ABA in seed germination, seedling establishment and root 353

growth, we speculated that they might regulate some common genes involved in ABA 354

responses. Therefore, we performed RNA-seq experiments to profile the 355

transcriptome of ndx-4, atring1a atring1b and the wild type under different conditions. 356

We used ndx-4 because it is a T-DNA insertion mutant with a cleaner genetic 357

background than the EMS mutants (ndx-5 and ndx-6). Total RNAs were isolated from 358

7-d-old seedlings treated with or without ABA for 3 h and used for mRNA-seq library 359

construction. We sequenced two biological replicates for each sample on an Illumina 360

NovaSeq 6000 platform and obtained more than 30 million paired-end clean reads for 361

each replicate. RNA-seq reads were mapped back to the TAIR10 genome, and gene 362

expression levels were quantified using the software cuffdiff (Trapnell et al., 2013). 363

Differentially expressed genes (DEGs) were filtered with FDR < 0.05 and fold 364

change >2 and required to have an expression higher than 1 FPKM (Trapnell et al., 365

2013). 366

Under normal conditions (i.e., no ABA treatment), 384 and 1798 genes were 367

upregulated in ndx-4 and atring1a atring1b, respectively, while 54 and 1409 genes 368

12

were downregulated in ndx-4 and atring1a atring1b, respectively, compared with the 369

wild type (Figure 5A, Supplemental Data Set 1, Sheet 1-11). Among these genes, 252 370

were co-upregulated and 22 were co-downregulated in the two mutants (Figure 5A). 371

We classified the genes co-regulated by AtNDX and AtRING1A/B under normal 372

conditions and found that these genes encode proteins with various functions, most of 373

them annotated as stimulus/stress responses and developmental processes (Figure 5B). 374

Under ABA treatment, 279 and 1467 genes were upregulated while 73 and 2382 genes 375

were downregulated in ndx-4 and atring1a atring1b, respectively, compared with the 376

wild type (Figure 5C). Among them, 163 genes were co-upregulated and 36 genes 377

were co-downregulated in the two mutants (Figure 5C). 378

To identify the genes that lead to ABA-hypersensitive phenotypes in the ndx and 379

atring1a atring1b mutants, we examined the overlap between the co-regulated genes 380

in the two mutants under normal conditions and the genes regulated by ABA signaling 381

in the wild type. In the wild type, 2269 genes were ABA-induced and 2774 genes 382

were ABA-repressed. Among the total 279 co-regulated genes in ndx-4 and atring1a 383

atring1b under normal conditions, 105 genes were responsive to ABA in the wild type, 384

including 80 ABA-induced genes and 25 ABA-repressed genes (Figure 5D, 385

Supplemental Data Set 1, Sheet 12). We then compared the expression levels of these 386

co-regulated and ABA-responsive genes under normal and ABA treatment conditions. 387

Heat map analysis indicated that the expression levels of most of these co-regulated 388

genes in ndx-4 and atring1a atring1b were higher than those in the wild type under 389

both normal conditions and ABA treatment (Figure 5E). 390

We used reverse transcription-quantitative PCR (RT-qPCR) to confirm the 391

expression of a sample of genes, including PLT5 (PLETHORA5), SUT4 (SUCROSE 392

TRANSPORTER4), CHI (CHALCONE ISOMERASE) and EM1 (LATE 393

EMBRYOGENESIS ABUNDANT1) from those that were both co-regulated and 394

ABA-responsive, and AT3G63410 from those that were neither co-regulated nor 395

ABA-responsive and found the results to be consistent with the RNA-seq data. SUP 396

(SUPERMAN) served as a control for no change in expression (Figure 5F). We 397

checked the expression levels of several major components in the ABA signaling 398

pathway and found that ABI3, ABI4, and ABI5 displayed much higher expression in 399

atring1a atring1b than in the wild type (Figure 5F), but were not identified by 400

RNA-seq owing to their low expression (ABI3, ABI4) or their insignificant difference 401

13

in expression between ndx-4 and wild type (ABI5). The RT-qPCR results indicated 402

that ABI3 and ABI4 were also expressed at higher levels in ndx-4 than in the wild type 403

in presence of ABA, but ABI5 was not (Figure 5F), suggesting that the expression of 404

ABI3 and ABI4 is modulated by AtNDX. 405

AtNDX is required for normal H2Aub levels in ABI4 and other ABA-responsive 406

genes 407

As core components of PRC1, AtRING1A/B mediate histone H2A 408

monoubiquitination (H2Aub) in vitro and in vivo (Bratzel et al., 2010; Li et al., 2017). 409

As AtNDX interacts with AtRING1A/B, we wanted to know whether the H2Aub level 410

would be affected by AtNDX. We performed immunoblotting analysis using total 411

proteins extracted from 7-d-old seedlings. Interestingly, we found that the total 412

H2Aub level was significantly reduced in each of the three ndx mutants (Figure 6A 413

and 6B). Since AtNDX did not affect the expression of AtRING1A/B (Supplemental 414

Figure 6), these results suggest that AtNDX is required for H2Aub modification, 415

which most likely relies on protein interaction between AtNDX and AtRING1A/B. 416

We hypothesize that if PRC1 works with AtNDX to regulate its target genes, the 417

H2Aub on some loci should be altered in the ndx mutants. Referring to published data 418

(Zhou et al., 2017), we identified 93 H2Aub-marked genes, representing about 84% of 419

the 105 co-regulated and ABA-responsive genes (Figure 6C). We then selected some 420

of these genes (Supplemental Figure 9) and checked their H2Aub levels by a 421

chromatin immunoprecipitation (ChIP) assay. Among these genes, the H2Aub levels 422

of PLT5, SUT4, CHI, EM1, and ABI4 were significantly reduced in both ndx-4 and 423

atring1a atring1b compared with the wild type, while the H2Aub level of ABI3 was 424

reduced slightly but not significantly. The H2Aub levels of AT3G63410 and ABI5 425

were apparently unchanged (Figure 6D), which is consistent with the lack of 426

difference in their expression levels in ndx-4 and atring1a atring1b relative to the 427

wild type (Figure 5F). SUP (SUPERMAN) was used as a negative control without 428

H2Aub. Collectively, our results demonstrate that AtNDX and AtRING1A/B affect 429

H2Aub levels of ABI4 and some ABA-responsive genes. These results suggest that 430

that H2Aub levels are negatively correlated with the expression levels of some genes, 431

but they are not necessarily linearly related, which is similar to several repressive 432

histone modifications reported before (Huang et al., 2013). 433

ABI4 is a direct target of AtNDX 434

14

In order to find the possible targets of AtNDX in ABA-regulated root growth, we 435

performed genetic analyses of several major components in the ABA signaling 436

pathway. We found that the abi4-1 mutation could rescue the ABA-hypersensitive 437

phenotype of the ndx-5 mutant with respect to both primary root growth and seedling 438

establishment (Figure 7A–7D). However, abi4-1 single mutants showed similar ABA 439

sensitivity in primary root growth as the wild type (Figure 7A, 7B). This result 440

suggests that as a positive regulator, ABI4 level in the wild type is not high enough to 441

inhibit the primary root growth under ABA treatment, but the higher expression of 442

ABI4 in ndx-5 mutants leads to more sensitivity to ABA in primary root growth, 443

compared with in the wild type. 444

To further confirm the results, we created two CRISPR/Cas9 abi4 mutants in ndx-4 445

and ndx-5 using an egg-specific promoter system (Wang et al., 2015), among which a 446

chimeric protein was produced in abi4-C1 and a truncated protein in abi4-C2. The 447

ABA-sensitive phenotypes of ndx-4 abi4-C1 and ndx-5 abi4-C2 were similar to those 448

of the wild type (Supplemental Figure 10A–10D), confirming that ABI4 acts at or 449

downstream of AtNDX in ABA signaling. By contrast, ndx-5 abi1-1 (Col-0), ndx-5 450

abi2-1 (Ler) double mutants and the ndx-5 snrk2.2/2.3 triple mutant displayed 451

intermediate ABA-sensitive phenotypes, suggesting that AtNDX is one of the 452

downstream targets of these components in the early ABA signaling pathway 453

(Supplemental Figure 11A–11F). The ndx-5 abi3-1 and ndx-5 abi5 double mutants 454

displayed similar primary root growth phenotypes to ndx-5, but exhibited 455

ABA-insensitive seedling establishment phenotypes similar to those of abi3-1 and 456

abi5 (Supplemental Figure 11G–11L). These findings suggest that ABI3 and ABI5 act 457

at or downstream of AtNDX in seedling establishment, but play different roles in 458

ABA-inhibited primary root growth. Indeed, recently it was found that ABI5 459

mutations can suppress the primary root ABA-sensitive phenotype of the glk1 460

(golden2-like1) glk2 wrky40 triple mutant in Arabidopsis, suggesting that ABI5 also 461

functions in primary root growth, although like abi4, abi5 single mutant did not show 462

any primary root growth phenotype to ABA (Ahmad et al., 2019). 463

Although studies have reported diverse functions of ABI4, including regulating 464

lateral root growth (Shkolnik-Inbar and Bar-Zvi, 2010) and increasing ABA 465

accumulation, decreasing GA content, and causing dwarfing when overexpressed 466

(Shu et al., 2016), few studies have examined the role of ABI4 in regulating primary 467

15

root growth. To test this, we produced estradiol-inducible ABI4-overexpression 468

transgenic lines and examined their phenotype on ABA medium. High expression of 469

ABI4 induced by addition of estradiol inhibited primary root growth on MS medium, 470

which is consistent with previous results on ectopic, constitutive ABI4 expression 471

(Soderman et al., 2000). ABI4 overexpression also caused increased sensitivity to 472

ABA in terms of primary root growth (Figure 7E and 7F). These results suggest that 473

ABI4 plays a positive role in ABA inhibition of primary root growth. 474

The above results prompted us to test whether AtNDX directly targets ABI4. To 475

search for AtNDX targets, we performed ChIP-seq analysis using anti-NDX 476

antibodies or using AtNDX-GFP complemented ndx seedlings with anti-GFP 477

antibodies, but failed to pull down enough genomic DNA for further analysis, 478

probably because of low antibody quality or a low amount of AtNDX protein. We 479

then used DNA affinity purification sequencing (DAP-seq) to examine AtNDX 480

binding (O'Malley et al., 2016). AtNDX is a homeodomain (HD)-containing protein 481

that belongs to a large family of transcription factors with the HD preferably binding 482

to a core TAAT motif (Mukherjee et al., 2009; Christensen et al., 2012). The 483

homeodomain of AtNDX is atypical and highly divergent (Mukherjee et al., 2009). 484

AtNDX has been reported to bind the single-stranded-DNA at the 3′ region of FLC in 485

a non-sequence-specific manner (Sun et al., 2013). We used the GST-fused 486

C-terminus of AtNDX protein (including the HD and NDXB domain) to pull down 487

fragmented genomic DNA in vitro as we could not purify full-length AtNDX from E. 488

coli (Sun et al., 2013). The DAP-seq analysis revealed that the AtNDX fragment 489

preferred to bind the promoter and terminal region of genes and that the AtNDX 490

binding sequence usually possessed a high AT ratio (Supplemental Figure 12A and 491

12B). 492

We noticed that there were both weak and strong binding signals close to the 493

ABI3/4/5 genes (Figure 8A). We further examined whether these loci were targeted by 494

AtNDX in vivo by using a ChIP-PCR assay. Here we used the ndx-1 (eoc1-1) mutant 495

complemented by AtNDX driven by its native promoter and fused with GFP in frame 496

(Sun et al., 2013). 7-day-old seedlings were used for a ChIP-PCR assay with GFP 497

antibodies, and pro35S:GFP transgenic seedlings were used as a control. We found 498

that a significant enrichment was detected around the ABI4 downstream region 499

(around 730-1013 bp from the putative stop codon), but not in the promoter region 500

16

(Figure 8B). For ABI3 and ABI5, we did not detect a clear AtNDX enrichment (Figure 501

8C). 502

We then confirmed the binding in vitro through an electrophoretic mobility shift 503

assay (EMSA) at the DAP-seq peak localized in the ABI4 downstream region (Figure 504

8D and 8E). We noticed that AtNDX-GST could bind to both single-stranded DNA 505

(ssDNA) and double-stranded DNA (dsDNA) of this region, but had stronger binding 506

affinity to dsDNA (Figure 8E). This differs from the results of a previous study, in 507

which AtNDX only bound to ssDNA (Sun et al., 2013), probably due to the different 508

probes used in two assays (Sun et al., 2013). These results suggest that AtNDX can 509

bind both ssDNA and dsDNA. The GST negative control did not show any binding 510

affinity. We observed that either TATA or ATTA mutations decreased the binding 511

affinity (Figure 8E). Moreover, AtNDX binding affinity seemed to be related to the 512

AT content of the sequence (Figure 8E). 513

As AtNDX can bind the downstream region of ABI4 and repress its expression, 514

we wanted to know whether deletion of the ABI4 downstream fragment would affect 515

ABI4 expression. We used CRISPR/Cas9 to create two deletion mutants (abi4-T1, 516

abi4-T2) in the downstream region of ABI4 (Wang et al., 2015). The two 517

CRISPR/Cas9 mutants were more sensitive to ABA than the wild type, but less 518

sensitive to ABA than ndx-4 with respect to primary root growth (Figure 8G-8F). 519

Consistently, we found that the expression of ABI4 under ABA treatment was 520

significantly increased in these two CRISPR/Cas9 lines (Figure 8I). These results 521

suggest that the downstream region of ABI4 plays a negative role in regulation of 522

ABI4 expression. 523

DISCUSSION 524

In this report, we provide several lines of evidence showing that AtNDX interacts 525

with the PRC1 core components AtRING1A and AtRING1B, and that they 526

co-regulate the H2Aub modification and expression of some ABA-responsive genes 527

(Figure 3, Figure 5 and Figure 6). Interestingly, ABA negatively regulates the 528

expression of AtNDX. Under normal conditions, a larger number of AtNDX and 529

AtRING1A/B co-regulated ABA-responsive genes were up-regulated than 530

down-regulated in ndx-4 and atring1a atring1b mutants relative to wild type, 531

suggesting that AtNDX and AtRING1A/B are required for repressing these genes 532

17

under the normal conditions. AtNDX and AtRING1A/B mutants are hypersensitive to 533

ABA in seed germination, seedling establishment and root growth, but not in ABA 534

promoted stomatal closure, indicating that they are negative regulators in some 535

ABA-mediated responses. We found that in ndx mutants, the H2Aub level was 536

apparently reduced but the expression levels of AtRING1A and AtRING1B were 537

unchanged, suggesting that H2Aub modification is mediated by AtNDX at the 538

posttranscriptional level. Furthermore, most of the AtNDX and AtRING1A/B 539

co-regulated ABA-responsive genes have H2Aub modification. We found an apparent 540

reduction in H2Aub in these co-regulated ABA-responsive genes, including ABI4, in 541

the ndx and atring1a atring1b mutant. So we speculate that AtNDX might relate to 542

the AtRING1A/B’s function in modifying the target genes, such as ABI4. 543

Under drought stress conditions, the upstream components in the ABA signaling 544

pathway such as ABA receptors, SnRKs, and PP2Cs usually modulate both the 545

expression of ABA-responsive genes at the transcriptional level in a relatively slow 546

response and guard cell movement at the posttranscriptional level in a quick response 547

(Cutler et al., 2010; Zhu, 2016; Qi et al., 2018). Here we found that ndx mutants 548

exhibited ABA super-sensitivity with respect to seedling establishment and primary 549

root growth. Our genetic analyses with classic ABA-responsive mutants indicate that 550

ndx mutation compromised the ABA-insensitive phenotypes of abi1-1 (Col-0) (Hua et 551

al., 2012), abi2-1 (Leung et al., 1997) and snrk2.2/2.3 (Fujii et al., 2007) double 552

mutants, further suggesting that AtNDX is one of downstream targets in ABA 553

signaling (Supplemental Fig. 11). During the seedling establishment stage, the 554

ABA-insensitive phenotypes of abi3 (Giraudat et al., 1992), abi4 (Finkelstein et al., 555

1998) and abi5 (Finkelstein and Lynch, 2000) were not changed by introducing the 556

ndx mutation, suggesting that ABI3/4/5 function genetically downstream of AtNDX. 557

The expression of ABI3 is increased in ndx, but we did not find that AtNDX could 558

directly bind to ABI3 in the ChIP-PCR analysis, suggesting either that AtNDX does 559

not directly target ABI3 or that our technique is not sensitive enough to detect the 560

binding. Indeed, the regulation network of ABI3, ABI4, and ABI5 is very complex 561

(Soderman et al., 2000; Feng et al., 2014). 562

Although previous studies had shown diverse functions of ABI4 (Wind et al., 563

2013; Feng et al., 2014; Li et al., 2014; Shu et al., 2016; Huang et al., 2017), its role in 564

regulating primary root growth in an abi4 mutant had not been revealed. Our genetic 565

18

studies show that ABI4 mutations can rescue the ABA-hypersensitive seedling 566

establishment and primary root growth phenotypes of ndx mutants, and that 567

overexpression of ABI4 in an inducible manner retarded primary root growth and 568

increased sensitivity to ABA. These results suggest that ABI4 functions downstream 569

of AtNDX in regulating primary root growth. Given that abi4-1 does not show any 570

primary root growth difference comparing with the wild type under ABA treatment, 571

we speculate one reason is that the expression of ABI4 is precisely and strictly 572

regulated during plant development. In the wild type after germination, ABI4 is 573

gradually silenced and stays at a very low expression level that is not enough to 574

inhibit primary root growth under ABA treatment. However, in the ndx mutants and 575

ABI4 overexpression lines, ABI4 is expressed to reach a high level that is sufficient to 576

inhibit the primary root growth. Therefore, we think the targets of ABI4 are probably 577

directly related to ABA response. 578

In our study, we found by DAP-seq and EMSA that AtNDX is able to bind the 579

downstream region of ABI4, while H2Aub is mainly distributed in the ABI4 gene body, 580

especially in the first exon (Zhou et al., 2017). How then does AtNDX promote 581

H2Aub modification and repress ABI4 expression? We speculate that AtNDX might 582

bind the downstream region of ABI4 and then form a DNA loop, allowing it to interact 583

with AtRNG1A/B in the coding region, promoting H2Aub modification and 584

suppressing ABI4 expression (Figure 9). One of the examples is a recent study on the 585

expression of GLABRA1 (GL1), in which the KANADI1-TARGET OF EAT1 (TOE1) 586

complex binds the 3′ downstream region of GL1 and then forms a loop in the 587

promoter region, repressing GL1 expression and inhibiting trichome formation (Wang 588

et al., 2019). Genome-wide Hi-C (a genome-wide chromatin conformation capture 589

[3C]) analysis in Arabidopsis indicates that promoter regions with repressive histone 590

modification markers such as H3K27me3 show a strong tendency to form 591

conformational linkages over long distances in gene bodies (Liu et al., 2016). ABI4 is 592

also regulated by BPC-recruited PRC2 in the promoter, which mediates H3K27me3 593

modification (Mu et al., 2017). Some components of PRC1 could interact with those 594

of the PRC2 complex to form a large complex (Wang and Shen, 2018). It is possible 595

that AtNDX interacts with AtRING1A/B to form a DNA loop after it binds to the 596

downstream region of ABI4 (Figure 9). In this study, we attempted to test this 597

hypothesis by the 3C technique, but failed due to a lack of suitable restriction enzyme 598

19

recognition sites in this region. Nevertheless, the deletion by CRISPR/Cas9 of the 599

ABI4 downstream region where AtNDX most likely binds increased ABI4 expression 600

and ABA sensitivity. The deletion lines were more ABA-sensitive in primary root 601

growth than the wild type, but less ABA-sensitive than ndx, suggesting that AtNDX 602

not only works in the downstream region of ABI4, but also, for example, works 603

together with PRC1 in the ABI4 coding region. Without ABA treatment, the 604

expression of ABI4 in two deletion lines was not increased, but increased in ndx 605

mutant, suggesting that AtNDX is required for repressing ABI4 expression under 606

normal condition (Figure 8I). 607

In our study, it was very hard to obtain a stably overexpressed ABI4 transgenic 608

line in later generations, suggesting that ABI4 can be easily silenced, likely due to its 609

sequence in coding region. A previous study showed that AtNDX could stabilize the 610

R-loop structure of FLC (Sun et al., 2013), and the present study reveals a novel role 611

for AtNDX working with PRC1 for gene repression, suggesting that AtNDX uses 612

different regulatory mechanisms to control gene expression. When comparing gene 613

expression patterns, the number of genes co-regulated by AtNDX and 614

AtRING1A/AtRING1B was not very large; one possible explanation is that some 615

genes may be regulated by AtNDX-mediated R-loop formation during transcription. 616

Recent studies have indicated that, like other epigenetic markers such as histone 617

modification and DNA methylation, R-loops play crucial roles in various cellular 618

processes including gene expression and DNA repair (Xu et al., 2017; Zhou et al., 619

2017; Crossley et al., 2019). In order to fully understand the functions of AtNDX, it 620

will be necessary to perform genome-wide profiles of R-loops and H2Aub in ndx and 621

atring1a atring1b mutants. However, more improved techniques are needed as 622

AtNDX protein availability is a limiting factor: the protein is not highly accumulated 623

even when the gene is driven by the 35S promoter (this study) (Sun et al., 2013). This 624

may be also the possible reason for why the overexpressed AtNDX lines did not show 625

ABA insensitivity while only rescued the ndx mutant phenotype. 626

Many studies have shown that epigenetic regulation is involved in plant abiotic 627

stress responses (Chinnusamy et al., 2008; Yuan et al., 2013). A recent study indicates 628

that PRC2 attenuates ABA-induced senescence in Arabidopsis in a long-term process 629

(Liu et al., 2018a). The PRC1 complex also represses the expression of some 630

important genes in phase transitions during different developmental stages (Calonje, 631

20

2014; Wang and Shen, 2018). The connection between ABA signaling and chromatin 632

modification has been explored in several previous studies (Chinnusamy et al., 2008; 633

Saez et al., 2008; Han et al., 2012; Peirats-Llobet et al., 2016; Zhao et al., 2017; Liu et 634

al., 2018b). For example, SWI3B, a subunit of the SWI/SNF chromatin-remodeling 635

complex, interacts with HYPERSENSITIVE TO ABA1 (HAB1) and positively 636

modulates ABA signaling (Saez et al., 2008). The SWI/SNF ATPase BRAHMA 637

(BRM) can directly bind to the transcription start site of ABI5 and maintain the 638

well-positioned nucleosome for repressing the expression of ABI5 (Han et al., 2012). 639

The activity of BRM is adversely regulated by phosphorylation and 640

dephosphorylation mediated by SnRK2s and PP2CA (Peirats-Llobet et al., 2016), 641

which would result in activation of ABI5 or maintaining the repression status of ABI5, 642

respectively. H3K4me3 and H3K27me3 marks were found to be involved in drought 643

stress memory (Liu et al., 2014). ARABIDOPSIS TRITHORAX4 (ATX4) and ATX5 644

directly regulate the expression of ABA-HYPERSENSITIVE GERMINATION 3 645

(encoding a PP2C) through H3K4 methylation by binding at the locus (Liu et al., 646

2018b). The finding in this study that AtNDX interacts with PRC1 core components 647

AtRING1A/B to mediate ABA signaling provides novel evidence for the direct 648

connection between ABA signaling and epigenetic regulation. 649

MATERIALS AND METHODS 650

Plant materials and growth conditions 651

The Arabidopsis thaliana seeds were surface-sterilized and sown on MS medium 652

containing 2% sucrose and 0.8% or 0.9% agar. After stratified at 4oC for 3 days, the 653

plates were transferred to a light incubator (PHILIPS F17T8/TL841 bulb) with a light 654

intensity of 60 μE m−2

s−1

under 22-h-light/2-h-dark at 22oC. The mutants used in this 655

study Atring1a, Atring1b-2 (Shen et al., 2014), Atring1a-/-

Atring1b+/-

(Xu and Shen, 656

2008), ring1a-2 ring1b-3 (Li et al., 2017), eoc1-4 is renamed as ndx-4 in this study 657

(Sun et al., 2013), abi1-1 (Kong et al., 2015), snrk2.2/2.3 (Wang et al., 2018), abi4-1 658

(Liu et al., 2010) were in the Col-0 background; abi2-1, abi3-1 (Wang et al., 2011) 659

were in the Landsberg erecta (Ler) background, abi5 were in the Wassileskija (Ws) 660

background. The transgenic plants AtNDX-FLAG (in eoc1-4, ndx-4 in this study) and 661

AtNDX-eGFP (in eoc1-1, ndx-1) renamed as proAtNDX: AtNDX-Flag and proAtNDX: 662

AtNDX-GFP were as described before (Sun et al., 2013). For transgenic plants 663

overexpressing AtNDX and AtRING1A, their CDSs were fused with the indicated tag 664

21

and driven by the super promoter (Ni et al., 1995) and cloned into the pCAMBIA1300 665

vector. For the β-estradiol inducible ABI4 overexpression lines, the coding sequence 666

of ABI4 was fused with GFP tag and cloned into the pER8 vector (Zuo et al., 2000). 667

The constructs were introduced into Agrobacterium tumefaciens strain GV3101 and 668

then transformed into the ndx-5 mutant or wild type by floral infiltration. The primers 669

used to examine these mutants and construct the transgenic plants are listed in 670

Supplemental Table 1. 671

To generate the ABI4 downstream deletion lines (abi4-T1 and abi4-T2), targeting the 672

fragment from 618 bp and 1007 bp downstream of ABI4 (counting after the ABI4 673

putative stop codon) was selected for CRISPR/Cas9 editing. The targets were cloned 674

into the pHSE401 vector as described previously (Wang et al., 2015) and the 675

constructs were transformed into Col-0 by floral dip. The transgenic T1 seeds were 676

screened on MS medium containing 25 mg/L hygromycin. To identify the deletion 677

lines, about 80 T1 plants were first screened by PCR then verified by sequencing, and 678

the homozygous mutants were used to generate T2. To generate ndx-4 abi4-C1 and 679

ndx-5 abi4-C2 mutants, the same method was used, except that targets at the coding 680

sequence of ABI4 were selected and the constructs were introduced into ndx-4 and 681

ndx-5 mutant. T2 plants were harvested individually, and T3 seeds were screened on 682

hygromycin medium for the non-hygromycin-resistant lines. The primers used to 683

examine these mutants, to construct the CRISPR/Cas9 vector and transgenic plants 684

are listed in Supplemental Tables 2–4. 685

Map-based cloning of AtNDX 686

The mutants ndx-5 and ndx-6 were isolated from approximately 20,000 EMS- (ethyl 687

methane sulfonate) mutated M2 seedlings based on the root bending assay. ndx-5 688

mutant plants were crossed with Landsberg erecta plants. A total of about 1,300 ndx-5 689

mutant plants were selected from the self-fertilized F2 population based on the 690

ABA-hypersensitive phenotype on 30 μM ABA. The simple sequence-length 691

polymorphism (SSLP) markers were used for mapping. The position of the AtNDX 692

mutation was determined within BAC clone T4I9. All genes in the BAC clone T4I9 693

were sequenced, and the mutation in the At4g03090 gene was identified in ndx-5. 694

ndx-6 was mapped to the same BAC clone as ndx-5, and a mutation in ndx-6 was 695

found in At4g03090. For the complementation, an 8 kb fragment that included about 696

3000 bp upstream of the first putative ATG to 1000 bp downstream of the putative 697

22

stop codon TGA of At4g03090 gene was amplified from genomic DNA and cloned 698

into pCAMBIA1391 vector. The constructs were introduced into Agrobacterium 699

tumefaciens strain GV3101 and then transformed into the ndx-5 mutant or wild type 700

by floral infiltration. The several independent transgenic lines obtained were able to 701

complement the ABA-hypersensitive phenotypes of ndx-5. 702

ABA-related phenotype analyses 703

For the root growth assay, seeds were germinated on the vertically oriented plates, 704

and 5-d-old seedlings were transferred to MS medium supplemented with different 705

concentrations of ABA for 3 or 4 days. Then the seedlings were photographed and the 706

root growth after transfer (below the dotted line) was measured by Image J. For the 707

calculation of relative root growth, the root growth of each genotype on MS medium 708

without ABA was set to 100%. For the seed germination greening ratio assay, seeds 709

were sown on MS medium supplemented with different concentrations of ABA and 710

grew for 2-11 days after sowing before photographed, and the ratio of seedlings with 711

ruptured endosperm, emerged radical and expended green cotyledons was calculated. 712

Observation of root meristem and mature epidermal cell 713

To measure of meristem cell number, a drop of transparent liquid was added to a glass 714

slide, and the root tips of the seedlings were immersed in the transparent liquid, then 715

covered with a glass cover and placed under a microscope (Olympus BX53). The cells 716

in the meristem were observed with a 20-fold objective lens and photographed. The 717

transparent liquid contains 60 mL deionized water, 7.5 g Gum Arabic, 100 g hydrated 718

trichloroacetaldehyde and 5 mL Glycerol. 719

To measure the length of mature epidermal cell, roots were treated as following steps: 720

(1) aqueous solution containing 4% HCl and 20% methanol at 57 °C for 15 min; (2)721

aqueous solution containing 7% NaOH and 60% ethanol at room temperature for 15 722

min; (3) 60% ethanol solution at room temperature for 10 min; (4) 40% ethanol 723

solution at room temperature for 10 min; (5) 20% ethanol solution at room 724

temperature for 10 min; (6) 10% ethanol solution at room temperature for 10 min; (7) 725

aqueous solution containing 25% glycerol and 5% ethanol at room temperature for 10 726

min; (8) store in 50% glycerol solution and observe the film. The films were observed 727

under a 10-fold objective lens and photographed, and the cell length was measured 728

using ImageJ software. For each root, the first five epidermal cells (near to the distal) 729

23

from the first cell with root hair were measured and used to calculate the average 730

length. 731

Physiological Experiments 732

For the water loss in detached leaves, shoots were cut from plants growing under 733

normal conditions for 4 weeks, and placed on a piece of weighing paper under a light 734

in a greenhouse, weighed immediately, every half an hour or one hour. The results are 735

shown as a percentage of the fresh weight. More than two independent experiments 736

were performed, each including three or four replicate shoots per line. 737

For stomatal density, epidermal strips were peeled from 4-weeks plants and the 738

mesophyll cells were removed with a small brush. The cleaned epidermal strips were 739

photographed under a microscope (Olympus BX53) with a 40-fold objective lens and 740

photographed. The stomata numbers were counted. 741

GUS staining 742

A 1609-bp fragment upstream from the start codon ATG of AtNDX was amplified by 743

PCR. The amplified fragment was cloned into the pCAMBIA1391 vector for a 744

transcriptional fusion of the AtNDX promoter with the GUS coding region. 7-d-old 745

homozygous transgenic plants carrying proAtNDX:GUS were treated with 0 μM or 30 746

μM ABA for 12 hours, and incubated in GUS staining buffer (0.1M PBS, 0.5 mM 747

K4Fe(CN)6, 0.5 mM K3Fe(CN)6, 1 mg/mL X-gluc, 0.1% Triton X-100) for 6 hours at 748

37 ºC. 749

Fluorescence microscopy 750

5-d-old proAtNDX:AtNDX-GFP transgenic seedlings grown on MS medium were 751

transferred to MS medium supplemented with 0 μM or 30 μM ABA for 12 hours. 752

Then seedlings were incubated in 10 μM propidium iodide for 3 min (to visualize the 753

cell) before imaged with the Carl Zeiss LSM710 confocal microscope. The roots were 754

photographed under the same setting and the fluorescence intensities of a same area in 755

the root meristem zone were measured by ImageJ software. 756

Yeast two-hybrid assay 757

For yeast two-hybrid screening, a normalized universal Arabidopsis cDNA library 758

(Clontech, 630487) was screened with the full length of AtNDX as bait. The 759

screening was performed according to the operating instruction. For construction the 760

24

bait and prey, the full length or truncated AtNDX and AtRING1A/B were fused into 761

pGBKT7 (BD) or pGADT7 (AD) vectors. The plasmids were co-transfected into 762

yeast strain AH109. Then the transfected yeast cells were plated on SD/-Leu/-Trp 763

medium (-LW) and SD/-Ade/-His/-Leu/-Trp medium (-LWHA) and cultured at 30oC 764

for 5 days. The primers used for vector construction are listed in Supplemental Table 765

3. 766

Co-IP and LC-MS/MS Assays 767

The Co-IP assays were performed as described previously (Wang et al., 2018). Briefly, 768

the total proteins of Arabidopsis protoplasts transfected with pro35S:HA-Flag-AtNDX 769

and proSuper:AtRING1A-GFP or proSuper:AtRING1B-GFP, or transgenic plants 770

harboring proSuper:AtRING1A-GFP were extracted and immunoprecipitated by GFP 771

beads (gta-20, ChromoTek). The primers used for vector construction are listed in 772

Supplemental Table 3. The immunoprecipitated proteins were detected with indicated 773

antibodies by immunoblotting analysis. The antibodies used were anti-GFP antibodies 774

(ab290, Abcom), anti-HA antibodies (H6908, Sigma-Aldrich), anti-NDX antibodies 775

(produced by Shanghai Youke Biotechnology Co., Ltd. ). Anti-NDX antibodies are 776

polyclonal antibodies prepared by immunizing rabbits with a peptide comprising the 777

589th to 879th amino acid of the AtNDX protein. For LC-MS/MS, total proteins were 778

extracted from wild-type and ndx-5 mutant 10-d-old seedlings, and 779

immunoprecipitated with anti-NDX antibodies, and the precipitated products were 780

subjected to LC-MS/MS analysis as described previously (Kong et al., 2015). 781

RNA-seq analyses. 782

7-d-old seedlings were treated with 1/2 liquid MS medium supplemented with 0 or 60 783

μM ABA for 3 hours. The total RNAs were extracted using RNeasy Plant Mini Kit 784

(Qiagen, 74104) and sent to the Berry Genomics Corporation for library construction 785

and sequencing. The sequencing was performed on the Illumina Novaseq6000 786

platform. Each sample had two biological replicates and obtained about 12G clean 787

bases each replicate. RNA-seq reads were collapsed into non-redundant reads and 788

mapped back to TAIR10 genome with Araport 11 annotation using STAR aligner with 789

maximum 8 mismatches per pair reads (Dobin et al., 2013). The gene expression level 790

was quantified using the available software cuffdiff. The differential expression genes 791

(DEGs) were filtered with FDR < 0.05 and Fold change >2 and required to have an 792

25

expression higher than 1 FPKM (Trapnell et al., 2013). The Venn diagrams were 793

drawn on the website http://bioinformatics.psb.ugent.be/webtools/Venn/ and the E 794

values were caculated through hypergeometric test by R package. The heatmap was 795

drawn based on the expression levels of indicated genes by the software Mev 4.9.0 796

with default parameters (Saeed et al., 2003). The raw data have been deposited in the 797

NCBI Sequence Read Archive and are accessible through SRA accession code 798

PRJNA556351 (https://www.ncbi.nlm.nih.gov/sra/PRJNA556351). 799

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) 800

7-d-old seedlings were treated with different concentrations of ABA for 3 hours, and 801

the total RNAs were extracted by HiPure Plant RNA Mini Kit (Magen, R4151). 4-μg 802

total RNAs were used for first-strand cDNA synthesis by Maxima H Minus First 803

Strand cDNA Synthesis Kit (Theeermo Scientific, K1682). RT-qPCR was performed 804

as described previously (Kong et al., 2015). The primers used for RT-qPCR were 805

listed in Supplemental Table 5. 806

Chromatin immunoprecipitation (ChIP) 807

The ChIP assays were performed as described previously (Zhang et al., 2016) with a 808

little modification. For H2Aub ChIP, 10-d-old seedlings were ground into fine 809

powder and cross-linked with 1% formaldehyde for 10 min. The nuclei were isolated 810

and the chromatin was sonicated by Bioruptor (Diagenode) and immunoprecipitated 811

by H2Aub antibody (Cell Signaling, D27C4) and Protein A+G Magnetic Beads 812

(Millipore, 16-663). For AtNDX-GFP ChIP, 7-d-old proAtNDX: AtNDX-GFP/ndx-1 813

and pro35S: GFP/Col-0 transgenic plants were used and the chromatin was 814

immunoprecipitated by GFP beads (gta-20, ChromoTek). The precipitated DNAs 815

were recovered by ChIP DNA Clean & Concentrator kit (Zymo Research, D5205) and 816

analyzed by RT-qPCR. The primers used for ChIP-qPCR are listed in Supplemental 817

Table 6. 818

Purification of recombinant protein, electrophoretic mobility shift assay (EMSA) 819

and DNA Affinity purification sequencing (DAP-seq) 820

The DNA-binding and NDXB domain (1800-2634 bp) in the C-terminal of AtNDX 821

was fused in frame with GST in the pGEX-2T vector and expressed in E. coli BL21 822

(DE3). The fused protein was induced by 1 mM IPTG and incubated overnight at 16 823

ºC. The recombinant protein was purified with Glutathione Sepharose 4B (GE 824

26

Healthcare) following the instruction. EMSA was carried out using the LightShift™ 825

Chemiluminescent EMSA Kit (Thermo Scientific) according to the instructions. For 826

double strand probes, the 5’- biotin-labeled DNA primers were made by 20 min 100 827

oC denaturation and renaturation in room temperature, and purified by native 828

polyacrylamide gel electrophoresis and QIAEX II Gel Extraction Kit (QIAGEN). For 829

single strand probes, the primers were denatured for 20 min and then transferred to ice 830

immediately. Each 20-μL volume of binding reaction contained 10 fM Biotin-probe, 1 831

μg of protein. The probes were incubated with the GST-AtNDX-C protein at room 832

temperature for 30 min. The reaction products were separated by 6.5% native 833

polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane 834

and detected according to the instructions. The DAP-seq was performed according to 835

O'Malley et al. (O'Malley et al., 2016) with a little modification.The genomic DNA 836

was extracted from 7-d-old wild type seedlings and fragmented by Bioruptor 837

(Diagenode) to 200-500 bp. The GST-AtNDX-C protein was first incubated with 838

Glutathione Sepharose 4B beads for 2 h, and washed out the free protein. Then the 839

GST-AtNDX-C covered beads were incubated with 500 ng fragmented DNA for 1h. 840

After washed out the free DNA, the affinity purified DNA was eluted and used for 841

sequencing. Two repeats were sequenced and 5 M reads were obtained for each repeat. 842

The clean data were mapped to the Arabidopsis genome (TAIR10) using bowtie2 843

(Langmead and Salzberg, 2012) with the following parameters: -N 1 -p 5 -q; and the 844

NDX binding peaks were identified by MACS (version 1.4.2) software (Zhang et al., 845

2008). The raw data have been deposited in the NCBI Sequence Read Archive and are 846

accessible through SRA accession code PRJNA556351 847

(https://www.ncbi.nlm.nih.gov/sra/PRJNA556351). 848

849

Accession Numbers 850

Sequence data from this article can be found in the GenBank/EMBL data libraries 851

under the following accession numbers: AtNDX, AT4G03090; AtRING1A, 852

AT5G44280; AtRING1B, AT1G03770; AtBMI1A, AT2G30580; AtBMI1B, AT1G06770; 853

AtBMI1C, AT3G23060; AtEMF1, AT5G11530; LHP1, AT5G17690; PLT5, 854

AT5G57390 ; SUT4, AT1G09960; CHI, AT2G43570; EM1, AT3G51810; ABI3, 855

AT3G24650; ABI4, AT2G40220; ABI5, AT2G36270; ACT4, AT5G59370; SUP, 856

AT3G23130. Mutants used in this article can be obtained from the ABRC under the 857

27

following accession numbers: snrk2.2 (GABI-Kat 807G04), and snrk2.3 858

(SALK_107315). The RNA sequencing data and DAP sequencing data for this 859

research have been deposited in the NCBI Sequence Read Archive under accession 860

code PRJNA556351. 861

862

Supplemental Data 863

Supplemental Figure 1. Root cell division and elongation of ndx-5 mutant are 864

hypersensitive to ABA. Supports Figure 1. 865

Supplemental Figure 2. Seedling establishment and stomata movement analysis of 866

AtNDX. Supports Figure 1. 867

Supplemental Figure 3. Map-based cloning and mutation analysis of AtNDX. 868

Supports Figure 1. 869

Supplemental Figure 4. Complementation of ndx mutants. Supports Figure 1. 870

Supplemental Figure 5. AtNDX does not directly interact with other PRC1 871

components in yeast cells. Supports Figure 3. 872

Supplemental Figure 6. The transcriptional levels of AtNDX and AtRING1A/B in 873

corresponding mutants. Supports Figure 5. 874

Supplemental Figure 7. ndx-5 atring1a double mutant is more sensitive to ABA than 875

ndx-5 in seed germination but not in primary root growth. Supports Figure 5. 876

Supplemental Figure 8. ndx-5 atring1b-2 double mutant shows similar sensitivity 877

with ndx-5 to ABA in seedling establishment and primary root growth. Supports 878

Figure 5. 879

Supplemental Figure 9. ChIP-seq genome browser views from published results. 880

Supports Figure 6. 881

Supplemental Figure 10. Disruption of ABI4 by CRISPR/Cas9 rescues the 882

ABA-hypersensitive phenotype of ndx mutants. Supports Figure 7. 883

Supplemental Figure 11. Genetic analysis of AtNDX and ABA signaling pathway 884

members. Supports Figure 7. 885

Supplemental Figure 12. DAP-seq analysis shows that AtNDX prefers to bind the 886

28

promoter and terminal regions of genes that possess high AT ratio. Supports Figure 8. 887

888

Supplementary Tables 1-6. Primers used in this study. 889

Supplemental Data Set 1. Gene expression analysis from RNA-seq. Supports Figure 890

5. 891

892

893

ACKNOWLEDGMENTS 894

We thank Dr. Zhen Li at China Agricultural University for performing LC-MS/MS 895

analysis. We thank Dr. Lin Xu at Shanghai Institutes for Biological Sciences for 896

providing ring1a-/-

ring1b+/-

double mutants, Dr. Hao Yu at National University of897

Singapo for providing ring1a and ring1b-2 mutants, and Dr. Ligeng Ma at Capital 898

Normal University for providing ring1a-2 ring1b-3 double mutants. This work was 899

supported by grants from the National Major Project for Transgenic Organism 900

Breeding (Ministry of Agriculture and Rural Affairs of the People’s Republic of 901

China) (2016ZX08009002), Beijing Outstanding Univeristy Displine Program, and 902

National Science Foundation of China (31921001). 903

AUTHOR CONTRIBUTIONS 904

Z.G., and Y.Z. conceived and designed the experiments. Y.Z. performed most of905

experiments. X.H. analyzed the phenotypes of ABI4 deletion mutants and Y.D. 906

isolated the mutants and cloned AtNDX gene. S.L. performed the bioinformatic 907

analyses of RNA-seq and ChIP-seq data. Z.G., Y.Z. and X.H. wrote the manuscript. 908

All other authors participated in the discussion of the results and commented on the 909

manuscript. 910

911

912

29

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34

Figure 1. ndx mutants are hypersensitive to ABA in primary root growth and 1186

seedling establishment. 1187

(A) Primary root growth of ndx mutants is hypersensitive to ABA compared with the 1188

wild type. 5-d-old seedlings grown on MS medium were transferred to MS medium 1189

supplemented with 30 μM, 60 μM, 90 μM ABA for 4 days before photographed. Bar 1190

= 1 cm. The phenotype in 60 μM ABA was shown. ear1-1 served as a control. 1191

(B) Statistical analysis of relative root growth with different concentrations of ABA. 1192

The root growth of wild type and ndx mutants on MS medium without ABA was set 1193

to 100%. Error bars represent ± SE of fifteen seedlings from 3 plates in one 1194

representative experiment, and three independent experiments were done with similar 1195

results. 1196

(C) Seedling establishment of ndx mutants is hypersensitive to ABA. The seeds were 1197

germinated on MS medium supplemented with different concentrations of ABA for 9 1198

days before photographed. 1199

(D) The seedling greening ratio in (C). Error bars represent ± SE of about 90 seeds 1200

from three plates for one experiment, and three independent experiments were done 1201

with similar results. * represents significant difference compared with wild type, * P < 1202

0.05, ** P < 0.01, Student’s t-test. 1203

Figure 2. The expression of AtNDX is inhibited by ABA. 1204

(A) Relative transcript levels of AtNDX in 7-d-old wild type seedlings treated with 0 1205

μM or 30 μM ABA for 3 hours. Error bars represent ± SE of three technical replicates 1206

from one experiment, and three independent experiments were done with similar 1207

results. 1208

(B) Representative GUS staining results of proAtNDX:GUS transgenic plants treated 1209

with 0 μM or 30 μM ABA for 12 hours. 1210

(C) The fluorescence of AtNDX-GFP is reduced after ABA treatment. 5-d-old 1211

seedlings grown on MS medium were transferred to MS medium supplemented with 1212

30 μM ABA for 12 hours before observed. Bar = 50 μm. 1213

(D) Statistical analysis of the fluorescence of meristem zone in (C). The fluorescence 1214

was quantified by ImageJ software, and error bars represent ± SE of ten seedlings 1215

from 3 plates in one representative experiment, and three independent experiments 1216

35

were done with similar results. 1217

(E) Immunoblot assays indicate that AtNDX protein level is reduced after ABA1218

treatment. 7-d-old seedlings were treated by 0 μM (CK) or 50 μM ABA for 12 hours 1219

(top left), 100 μM CHX or 100 μM CHX +50 μM ABA for 12 hours (top right), 0 μM 1220

(CK) or 50 μM MG132 for 12 hours. Then the total proteins were extracted and 1221

detected with anti-NDX antibodies. GST-AtNDX-C purified from E. coli was used as 1222

a positive control. Actin protein serves as an loading control. The numbers indicate 1223

the relative gray value. 1224

Figure 3. NDX interacts with PRC1 core components AtRING1A and AtRING1B 1225

in vitro and in vivo. 1226

(A) AtNDX interacts with AtRING1A and AtRING1B in the yeast two-hybrid assay.1227

AD: Gal4 activation domain; BD: Gal4 DNA-binding domain. -LW: synthetic 1228

dropout medium without Leu and Trp; -LWHA: synthetic dropout medium without 1229

Leu, Trp, His and Ade. 1230

(B) Co-IP assay indicates that AtNDX interacts with AtRING1A and AtRING1B in1231

Arabidopsis protoplasts. The total proteins were isolated from protoplasts 1232

co-expressing HA-Flag-AtNDX and AtRING1A-GFP or AtRING1B-GFP proteins. 1233

Co-IP was carried out with GFP agarose beads and immunoblotting was conducted 1234

with anti-HA and anti-GFP antibodies. 1235

(C) Co-IP assay shows that AtNDX interacts with AtRING1A in two independent1236

transgenic lines. The total proteins were isolated from 10-day-old 1237

proSuper:AtRING1A-GFP transgenic seedlings (L1, L2) and Co-IP was carried out 1238

with GFP agarose beads. Immunoblotting was conducted with anti-NDX and 1239

anti-GFP antibodies. 1240

(D) Mass spectrometry analysis shows that AtRING1A co-purified with AtNDX. The1241

total proteins extracted from 10-d-old wild type and ndx-5 seedlings were 1242

immunoprecipitated with anti-NDX antibodies and the immunoprecipitated proteins 1243

were analyzed by LC-MS/MS. SC means sequence coverage of the protein. 1244

(E) The C-terminus of AtRING1A/B interacts with AtNDX in a yeast two-hybrid1245

assay. The structures of AtRING1A/B proteins and the diagrams of truncated proteins 1246

were shown on top. 1247

36

(F) The NDXA and NDXB domains of AtNDX are both required for interacting with1248

AtRING1A/B. The structure of AtNDX protein and the diagrams of truncated proteins 1249

were shown on top. 1250

Figure 4. The atring1a atring1b double mutants are hypersensitive to ABA in 1251

seedling establishment and primary root growth. 1252

(A) Primary root growth of atring1a atring1b (1a 1b) double mutant is hypersensitive1253

to ABA compared with the wild type. 4-d-old seedlings grown on MS medium were 1254

transferred to MS medium supplemented with 30 μM ABA for 3 days before 1255

photographed. Bar = 1 cm. 1256

(B) Statistical analysis of primary root growth in (A). Error bars represent ± SE of1257

forty five seedlings from three biological repeats. 1258

(C) Statistical analysis of relative root growth in (A). The root growth of wild type1259

and double mutant on MS medium without ABA was set to 100%. Error bars 1260

represent ± SE of forty five seedlings from three biological repeats. 1261

(D) Primary root growth of atring1a-2 atring1b-3 (1a-2 1b-3) double mutant is1262

hypersensitive to ABA compared with the wild type. 4-d-old seedlings grown on MS 1263

medium were transferred to MS medium supplemented with 60 μM ABA for 4 days 1264

before photographed. Bar = 1 cm. 1265

(E) Statistical analysis of relative root growth in different concentrations of ABA. The1266

primary root growth of the wild type and atring1a-2 atring1b-3 double mutant (1a-2 1267

1b-3) on MS medium without ABA was set to 100%. ear1-1 served as the positive 1268

control. Error bars represent ± SE of ten seedlings from one representative experiment, 1269

and three independent experiments were done with similar results. 1270

(F) Seedling establishment of atring1a-2 atring1b-3 double mutant (1a-2 1b-3) is1271

hypersensitive to ABA. The seeds were germinated on MS medium supplemented 1272

with 0.5 μM ABA for 9 days before photographed. 1273

(G) The seedling greening ratio in (F). Error bars represent ± SE of about 90 seeds1274

from three plates for one experiment, and three independent experiments were done 1275

with similar results. * Represents significant difference compared with the wild type, 1276

** P < 0.01, Student’s t-test. 1277

Figure 5. AtNDX and AtRING1A/B co-regulate ABI4 and a series of 1278

37

ABA-responsive genes. 1279

(A) Venn diagram shows the number of overlapped up- and down-regulated DEGs in 1280

ndx-4 and atring1a atring1b double mutant (1a 1b) compared with the wild type 1281

under control conditions. 1282

(B) GO annotation analysis of AtNDX and AtRING1A/B co-regulated genes. The 1283

abscissa represents a part of GO terms with significant enrichment, the ordinate 1284

represents the ratio of the genes enriched in the GO term to the total number of genes, 1285

and the number represents the E value of the significance analysis compared to the 1286

reference genome (TAIR10, 2017). 1287

(C) Venn diagram shows the number of overlapped up- and down-regulated DEGs in 1288

ndx-4 and atring1a atring1b double mutant (1a 1b) compared with the wild type 1289

under ABA treatment conditions. 7-d-old seedlings were treated with 0 or 60 μM 1290

ABA for 3 hours. 1291

(D) Venn diagram shows the overlapped number of co-up- and down-regulated genes 1292

under normal condition in ndx-4 and atring1a atring1b double mutant (1a 1b), which 1293

are ABA responsive genes in the wild type. 1294

(E) Heat map shows that the ABA responsive genes co-regulated by AtNDX and 1295

AtRING1A/B in (D) were mainly up-regulated in the ndx-4 and atring1a atring1b 1296

double mutant. The unit of expression is FPKM. 1297

(F) Relative expression level analyses of ABA related genes in the ndx-4 and atring1a 1298

atring1b double mutant (1a 1b). For each sample, 7-day-old seedlings were treated 1299

with 0 μM ABA (Control) or 60 μM ABA (ABA) for 3 hours, then the total RNAs 1300

were extracted. Error bars represent ± SE of three technical replicates from one 1301

experiment, and three independent experiments were done with similar results. * 1302

Represents significant difference compared with wild type, ** P < 0.01, Student’s 1303

t-test. 1304

Figure 6. AtNDX and AtRING1A/B regulate H2Aub levels of ABI4 and 1305

ABA-responsive genes. 1306

(A) Immunoblot assay indicates that the H2Aub levels in ndx mutants are reduced 1307

compared to the wild type. The nuclear proteins of 10-d-old seedlings were extracted 1308

and detected with H2Aub antibody, and H3 was used as an internal control. 1309

38

(B) Statistical analysis of relative band intensity in (A). The band intensity was 1310

quantified by ImageJ software, and the ratio of H2Aub to H3 in wild type was set to 1. 1311

Error bars represent ± SE of three biological replicates. ** P < 0.01, Student’s t-test. 1312

(C) Venn diagram shows the number of overlapped genes between AtNDX and 1313

AtRING1A/B co-regulated ABA responsive genes and H2Aub marked genes. 1314

(D) ChIP-qPCR analysis shows that the H2Aub levels of ABI4 and ABA responsive 1315

genes are reduced in ndx-5 and atring1a atring1b double mutant (1a 1b). The checked 1316

regions are labeled with the red lines below the gene diagrams in Supplemental Figure 1317

9. Error bars represent ± SE of three independent experiments. * Represent significant 1318

difference compared with wild type, *P < 0.05, **P < 0.01, Student’s t-test. 1319

Figure 7. ABI4 mutations rescue the ABA hypersensitive phonotype of ndx-5 and 1320

ABI4 overexpressing transgenic lines display ABA hypersensitive phonotype. 1321

(A) ndx-5 abi4-1 double mutant rescues the ABA hypersensitive phenotype of ndx-5 1322

in root growth. 5-d-old seedlings grown on MS medium were transferred to MS 1323

medium supplemented with 30 μM ABA for 4 days before photographed. Bar = 1 cm. 1324

(B) Statistical analysis of relative root growth with different concentrations of ABA 1325

treatment. The root growth of each genotype on MS medium without ABA was set to 1326

100%. Error bars represent ± SE of fifteen seedlings from one representative 1327

experiment, and three independent experiments were done with similar results. 1328

(C) ndx-5 abi4-1 double mutant rescues the ABA hypersensitive phenotype of ndx-5 1329

in seedling establishment. The seeds were germinated on MS medium supplemented 1330

with 0 or 0.5 μM ABA for 9 days before photographed. 1331

(D) The seedling greening ratio in (C). Error bars represent ± SE of about 90 seeds 1332

from three plates in one experiment, and three independent experiments were done 1333

with similar results. **P < 0.01, Student’s t-test. 1334

(E) Overexpression of ABI4 inhibits the root growth and confers ABA 1335

hypersensitivity. 5-days-old wild type and the transgenic wild type plants with 1336

inducible ABI4 overexpression were transferred to MS medium supplemented with 0 1337

or 30 μM ABA and without or with estradiol for 3 days before photographed. Bar = 1 1338

cm. 1339

(F) Statistical analysis of relative root growth in (E). The root growth of each 1340

39

genotype on MS medium without ABA and estradiol was set to 100%. Error bars 1341

represent ± SE of ten seedlings from three plates for one representative experiment, 1342

and three independent experiments were done with similar results. **P < 0.01, 1343

Student’s t-test. 1344

Figure 8. ABI4 is a direct target of AtNDX. 1345

(A) DAP-seq genome browser views show AtNDX binding signals around ABI3/4/5 1346

genes. 1347

(B) and (C) ChIP-qPCR analyses show that AtNDX binds to the ABI4 downstream 1348

sequence in vivo. The letters in (A) indicate the tested regions. DNA fragments 1349

immunoprecipitated by GFP beads were quantified by qPCR and normalized to the 1350

internal control ACT4; the relative enrichment in AtNDX-GFP over GFP is presented. 1351

Error bars represent ± SE of three technical replicates from one experiment, and three 1352

independent experiments were done with similar results. * Represent significant 1353

difference, **P < 0.01, Student’s t-test. 1354

(D) The probes used in EMSA. The sequence of ABI4-c-F probe is from the center of 1355

ABI4-c region in (A). The red letter represent TATA motif; the blue letters represent 1356

ATTA motif; mut1 and mut2 probes show the corresponding mutation sequence; F, 1357

forward strand DNA. 1358

(E) EMSA shows that recombinant GST-AtNDX-C binds to ABI4 downstream 1359

sequence in vitro. The upper and lower arrows indicate the bound probe and the free 1360

probe, respectively; ds, double-stranded DNA, indicates a double-stranded probe 1361

formed by annealing; F and R represent forward and reverse single-stranded probes. 1362

GST protein was used as a negative control. 1363

(F) The diagram of the binding peak of ABI4 downstream by AtNDX in DAP-seq and 1364

target location of CRISPR/Cas9. The sequence alignments show that the fragments 1365

from ABI4 downstream bases 624 to 1000 (counting after the first base of stop codon 1366

in ABI4) in abi4-T1 and the bases 605 to 968 in abi4-T2 were deleted, and there are 1367

insert mutant in target 2 in abi4-T2. These mutations almost cover the whole binding 1368

peak sequence of AtNDX. 1369

(G) abi4-T1 and abi4-T2 lines confer more ABA sensitivity than the wild type, but 1370

less ABA sensitive than ndx-4. 4-d-old seedlings grown on MS medium were 1371

transferred to MS medium supplemented 0 or 30 μM ABA for 4 days before 1372

40

photographed. Bar = 1 cm. 1373

(H) Statistical analysis of relative root growth in (G). The root growth of each1374

genotype on MS medium without ABA was set to 100%. Error bars represent ± SE of 1375

twelve seedlings from three plates for one representative experiment, and three 1376

independent experiments were done with similar results. **P < 0.01, Student’s t-test. 1377

(I) Relative transcript levels of ABI4 in 8-d-old seedlings. Seedlings were grown on1378

MS medium for 4 day, then transferred to MS medium supplemented 0 or 30 μM 1379

ABA for 4 day before harvested. Error bars represent ± SE of three technical 1380

replicates. ** P<0.01, Student’s t-test. 1381

Figure 9. A proposed model for the role of AtNDX and AtRING1A/B in ABA 1382

signaling. 1383

Under normal conditions (-ABA), AtNDX binds to the downstream region of ABI4 1384

and interacts with AtRING1A/B in the coding region, which promotes H2Aub 1385

modification and suppresses ABI4 expression. Under stress condition (+ABA), the 1386

expression of AtNDX is repressed by ABA signaling, which results in activation of the 1387

ABI4 expression. 1388

1389

1390

Figure 1. ndx mutants are hypersensitive to ABA in primary root growth and seedling establishment. (A) Primary root growth of ndx mutants is hypersensitive to ABA compared with the wild type. 5-d-old seedlings grown onMS medium were transferred to MS medium supplemented with 30 μM, 60 μM, 90 μM ABA for 4 days before photographed.Bar = 1 cm. The phenotype in 60 μM ABA was shown.ear1-1 served as the positive control.(B) Statistical analysis of relative root growth with different concentrations of ABA. The root growth of wild type and ndxmutants on MS medium without ABA was set to 100%. Error bars represent ± SE of fifteen seedlings from 3 plates in onerepresentative experiment, and three independent experiments were done with similar results.(C) Seedling establishment of ndx mutants is hypersensitive to ABA. The seeds were germinated on MS mediumsupplemented with different concentrations of ABA for 9 days before photographed.(D) The seedling greening ratio in (C). Error bars represent ± SE of about 90 seeds from three plates for one experiment,and three independent experiments were done with similar results. * represents significant difference compared with wildtype, * P < 0.05, ** P < 0.01, Student’s t-test.

Figure 2. The expression of AtNDX is inhibited by ABA. (A) Relative transcript levels of AtNDX in 7-d-old wild type seedlings treated with 0 μM or 30 μM ABA for 3 hours. Error barsrepresent ± SE of three technical replicates from one experiment, and three independent experiments were done withsimilar results.(B) Representative GUS staining results of proAtNDX:GUS transgenic plants treated with 0 μM or 30 μM ABA for 12 hours.(C) The fluorescence of AtNDX-GFP is reduced after ABA treatment. 5-d-old seedlings grown on MS medium weretransferred to MS medium supplemented with 30 μM ABA for 12 hours before observed. Bar = 50 μm.(D) Statistical analysis of the fluorescence of meristem zone in (C). The fluorescence was quantified by ImageJ software,and error bars represent ± SE of ten seedlings from 3 plates in one representative experiment, and three independentexperiments were done with similar results.(E) Immunoblot assays indicate that AtNDX protein level was reduced after ABA treatment. 7-d-old seedlings were treatedby 0 μM (CK) or 50 μM ABA for 12 hours (top left), 100 μM CHX or 100 μM CHX +50 μM ABA for 12 hours (top right), 0 μM(CK) or 50 μM MG132 for 12 hours. Then the total proteins were extracted and detected with anti-NDX antibodies.GST-AtNDX-C purified from E.coli was used as positive control. Actin proteinserves as an loading control. The numbersexpressed the relative gray value.

Figure 3. NDX interacts with PRC1 core components AtRING1A and AtRING1B in vitro and in vivo. (A) AtNDX interacts with AtRING1A and AtRING1B in the yeast two-hybrid assay. AD: Gal4 activation domain; BD: Gal4DNA-binding domain. -LW: synthetic dropout medium without Leu and Trp; -LWHA: synthetic dropout medium without Leu,Trp, His and Ade.(B) Co-IP assay indicates that AtNDX interacts with AtRING1A and AtRING1B in Arabidopsis protoplasts. The total proteinswere isolated from protoplasts co-expressing HA-Flag-AtNDX and AtRING1A-GFP or AtRING1B-GFP proteins. Co-IP wascarried out with GFP agarose beads and immunoblotting was conducted with anti-HA and anti-GFP antibodies.(C) Co-IP assay shows that AtNDX interacts with AtRING1A in two independent transgenic lines. The total proteins wereisolated from 10-day-old proSuper:AtRING1A-GFP transgenic seedlings (L1, L2) and Co-IP was carried out with GFPagarose beads. Immunoblotting was conducted with anti-NDX and anti-GFP antibodies.(D) Mass spectrometry analysis shows that AtRING1A co-purified with AtNDX. The total proteins extracted from 10-d-oldwild type and ndx-5 seedlings were immunoprecipitated with anti-NDX antibodies and the immunoprecipitated proteinswere analyzed by LC-MS/MS. SC means sequence coverage of the protein.(E) The C-terminus of AtRING1A/B interacts with AtNDX in a yeast two-hybrid assay. The structures of AtRING1A/Bproteins and the diagrams of truncated proteins were shown on top.(F) The NDXA and NDXB domains of AtNDX are both required for interacting with AtRING1A/B. The structure of AtNDXprotein and the diagrams of truncated proteins were shown on top.

Figure 4. The atring1a atring1b double mutants are hypersensitive to ABA in seedling establishment and primary root growth. (A) Primary root growth of atring1a atring1b (1a 1b) double mutant is hypersensitive to ABA compared with the wild type.4-d-old seedlings grown on MS medium were transferred to MS medium supplemented with 30 μM ABA for 3 days beforephotographed. Bar = 1 cm.(B) Statistical analysis of primary root growth in (A). Error bars represent ± SEM of forty five seedlings from three biologicalrepeats.(C) Statistical analysis of relative root growth in (A). The root growth of wild type and double mutant on MS medium withoutABA was set to 100%. Error bars represent ± SE of forty five seedlings from three biological repeats.(D) Primary root growth of atring1a-2 atring1b-3 (1a-2 1b-3) double mutant is hypersensitive to ABA compared with the wildtype. 4-d-old seedlings grown on MS medium were transferred to MS medium supplemented with 60 μM ABA for 4 daysbefore photographed. Bar = 1 cm.(E) Statistical analysis of relative root growth in different concentrations of ABA. The primary root growth of the wild typeand atring1a-2 atring1b-3 double mutant (1a-2 1b-3) on MS medium without ABA was set to 100%. ear1-1 served as thepositive control. Error bars represent ± SE of ten seedlings from one representative experiment, and three independentexperiments were done with similar results.(F) Seedling establishment of atring1a-2 atring1b-3 double mutant (1a-2 1b-3) is hypersensitive to ABA. The seeds weregerminated on MS medium supplemented with 0.5 μM ABA for 9 days before photographed.(G) The seedling greening ratio in (F). Error bars represent ± SE of about 90 seeds from three plates for one experiment,and three independent experiments were done with similar results. * Represents significant difference compared with thewild type, ** P < 0.01, Student’s t-test.

Figure 5. AtNDX and AtRING1A/B co-regulate ABI4 and a series of ABA-responsive genes. (A) Venn diagram shows the number of overlapped up- and down-regulated DEGs in ndx-4 and atring1a atring1b doublemutant (1a 1b) compared with the wild type under control conditions.(B) GO annotation analysis of AtNDX and AtRING1A/B co-regulated genes. The abscissa represents a part of GO termswith significant enrichment, the ordinate represents the ratio of the genes enriched in the GO term to the total number ofgenes, and the number represents the E value of the significance analysis compared to the reference genome (TAIR10,2017).(C) Venn diagram shows the number of overlapped up- and down-regulated DEGs in ndx-4 and atring1a atring1b doublemutant (1a 1b) compared with the wild type under ABA treatment conditions. 7-d-old seedlings were treated with 0 or 60μM ABA for 3 hours.(D) Venn diagram shows the overlapped number of co-up- and down-regulated genes under normal condition in ndx-4 andatring1a atring1b double mutant (1a 1b), which are ABA responsive genes in the wild type.(E) Heat map shows that the ABA responsive genes co-regulated by AtNDX and AtRING1A/B in (D) were mainlyup-regulated in the ndx-4 and atring1a atring1b double mutant. The unit of expression is FPKM.(F) Relative expression level analyses of ABA related genes in the ndx-4 and atring1a atring1b double mutant (1a 1b). Foreach sample, 7-day-old seedlings were treated with 0 μM ABA (Control) or 60 μM ABA (ABA) for 3 hours, then the totalRNAs were extracted. Error bars represent ± SE of three technical replicates from one experiment, and three independentexperiments were done with similar results. * Represents significant difference compared with wild type, ** P < 0.01,Student’s t-test.

Figure 6. AtNDX and AtRING1A/B regulate H2Aub levels of ABI4 and ABA-responsive genes. (A) Immunoblot assay indicates that the H2Aub levels in ndx mutants are reduced compared to the wild type. The nuclearproteins of 10-d-old seedlings were extracted and detected with H2Aub antibody, and H3 was used as an internal control.(B) Statistical analysis of relative band intensity in (A). The band intensity was quantified by ImageJ software, and the ratioof H2Aub to H3 in wild type was set to 1. Error bars represent ± SE of three biological replicates. ** P < 0.01, Student’st-test.(C) Venn diagram shows the number of overlapped genes between AtNDX and AtRING1A/B co-regulated ABA responsivegenes and H2Aub marked genes.(D) ChIP-qPCR analysis shows that the H2Aub levels of ABI4 and ABA responsive genes are reduced in ndx-5 and

atring1a atring1b double mutant (1a 1b). The red lines below the gene diagrams in Supplement Figure 8 indicate the checked regions. Error bars represent ± SE of three independent experiments. * Represent significant difference compared with wild type, *P < 0.05, **P < 0.01, Student’s t-test.

Figure 7. ABI4 mutations rescue the ABA hypersensitive phonotype of ndx-5 and ABI4 overexpressing transgenic lines display ABA hypersensitive phonotype. (A) ndx-5 abi4-1 double mutant rescues the ABA hypersensitive phenotype of ndx-5 in root growth. 5-d-old seedlingsgrown on MS medium were transferred to MS medium supplemented with 30 μM ABA for 4 days before photographed. Bar= 1 cm.(B) Statistical analysis of relative root growth with different concentrations of ABA treatment. The root growth of eachgenotype on MS medium without ABA was set to 100%. Error bars represent ± SE of fifteen seedlings from onerepresentative experiment, and three independent experiments were done with similar results.(C) ndx-5 abi4-1 double mutant rescues the ABA hypersensitive phenotype of ndx-5 in seedling establishment. The seedswere germinated on MS medium supplemented with 0 or 0.5 μM ABA for 9 days before photographed.(D) The seedling greening ratio in (C). Error bars represent ± SE of about 90 seeds from three plates in one experiment,and three independent experiments were done with similar results. **P < 0.01, Student’s t-test.(E) Overexpression of ABI4 inhibits the root growth and confers ABA hypersensitivity. 5-days-old wild type and thetransgenic wild type plants with inducible ABI4 overexpression were transferred to MS medium supplemented with 0 or 30μM ABA and without or with estradiol for 3 days before photographed. Bar = 1 cm.(F) Statistical analysis of relative root growth in (E). The root growth of each genotype on MS medium without ABA andestradiol was set to 100%. Error bars represent ± SE of ten seedlings from three plates for one representative experiment,and three independent experiments were done with similar results. **P < 0.01, Student’s t-test.

Figure 8. ABI4 is a direct target of AtNDX. (A) DAP-seq genome browser views show AtNDX binding signals around ABI3/4/5 genes.(B) and (C) ChIP-qPCR analyses show that AtNDX binds to the ABI4 downstream sequence in vivo. The letters in (A)indicate the tested regions. DNA fragments immunoprecipitated by GFP beads were quantified by qPCR and normalized tothe internal control ACT4; the relative enrichment in AtNDX-GFP over GFP is presented. Error bars represent ± SE of threetechnical replicates from one experiment, and three independent experiments were done with similar results. * Representsignificant difference, **P < 0.01, Student’s t-test. (D) The probes used in EMSA. The sequence of ABI4-c-F probe is fromthe center of ABI4-c region in (A). The red letter represent TATA motif; the blue letters represent ATTA motif; mut1 andmut2 probes show the corresponding mutation sequence; F, forward strand DNA.(E) EMSA shows that recombinant GST-AtNDX-C binds to ABI4 downstream sequence in vitro. The upper and lowerarrows indicate the bound probe and the free probe, respectively; ds, double-stranded DNA, indicates a double-strandedprobe formed by annealing; F and R represent forward and reverse single-stranded probes. GST protein was used as anegative control. (F) The diagram of the binding peak of ABI4 downstream by AtNDX in DAP-seq and target location ofCRISPR/Cas9. The sequence alignments show that the fragments from ABI4 downstream bases 624 to 1000 (countingafter the first base of stop codon in ABI4) in abi4-T1 and the bases 605 to 968 in abi4-T2 were deleted, and there are insertmutant in target 2 in abi4-T2 These mutation almost cover the whole binding peak sequence of AtNDX.(G) abi4-T1 and abi4-T2 lines confer more ABA sensitivity than the wild type, but less ABA sensitive than ndx-4. 4-d-oldseedlings grown on MS medium were transferred to MS medium supplemented 0 or 30 μM ABA for 4 days beforephotographed. Bar = 1 cm.(H) Statistical analysis of relative root growth in (G). The root growth of each genotype on MS medium without ABA was setto 100%. Error bars represent ± SE of twelve seedlings from three plates for one representative experiment, and threeindependent experiments were done with similar results. **P < 0.01, Student’s t-test.(I) Relative transcript levels of ABI4 in 8-d-old seedlings. Seedlings were grown on MS medium for 4 day, then transferredto MS medium supplemented 0 or 30 μM ABA for 4 day before harvested. Error bars represent ± SE of three technicalreplicates. ** P<0.01, Student’s t-test.

Figure 9. A proposed model for the role of AtNDX and AtRING1A/B in ABA signaling. Under normal conditions (-ABA), AtNDX binds to the downstream region of ABI4 and interacts with AtRING1A/B in the coding region, which promotes H2Aub modification and suppresses ABI4 expression. Under stress condition (+ABA), the expression of AtNDX is repressed by ABA signaling, which results in activation of the ABI4 expression.

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DOI 10.1105/tpc.19.00604; originally published online January 9, 2020;Plant Cell

Song, Qianwen Sun, Shuhua Yang and Zhizhong GongZhang, Deping Hua, Li Yang, Li Wang, Zhizhong Chen, Chuanyou Li, Baoshan Wang, Chun-Peng Yujuan Zhu, Xiaoying Hu, Ying Duan, Shaofang Li, Yu Wang, Amin Ur Rehman, Junna He, Jing

Regulates Abscisic Acid SignalingThe Arabidopsis Nodulin Homeobox Factor AtNDX Interacts with AtRING1A/B and Negatively

 This information is current as of February 12, 2020

 

Supplemental Data /content/suppl/2020/01/09/tpc.19.00604.DC1.html

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