regulation of female germline specification via small rna … · 2020. 7. 23. · germline...

1

RESEARCH ARTICLE 1 2

Regulation of Female Germline Specification via Small RNA Mobility 3

in Arabidopsis 4

5

Zhenxia Su1,2,3,4

, Nannan Wang4, Zhimin Hou

4, Baiyang Li

4，Dingning Li4, Yanhui Liu

4, 6

Hanyang Cai4, Yuan Qin

4,5*, Xuemei Chen

3,6* 7

8 1Guangdong Provincial Key Laboratory for Plant Epigenetics, Longhua Institute of 9 Innovative Biotechnology, College of Life Sciences and Oceanography, Shenzhen University, 10 Shenzhen 518060, China 11 2Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and 12 Guangdong Province, College of Optoelectronic Engineering, Shenzhen University, Shenzhen 13 518060, China 14 3Department of Botany and Plant Sciences, Institute of Integrative Genome Biology, 15 University of California, Riverside, California, 92521, United States 16 4Key Laboratory of Genetics, Breeding and Multiple Utilization of Crops, Ministry of 17 Education; Fujian Provincial Key Laboratory of Haixia Applied Plant Systems Biology, 18 Fujian Agriculture and Forestry University, Fuzhou 350002, China 19 5State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, 20 Guangxi Key Lab of Sugarcane Biology, College of Agriculture, Guangxi University, 21 Nanning 530004, Guangxi, China 22 6Lead Contact 23 *Correspondence: [email protected] or [email protected] 24

25 Short Title: Germline specification via small RNA mobility 26

27 One-sentence Summary: Intricate spatial restriction of tasiR-ARF biogenesis, together with 28 tasiR-ARF mobility, enables cell-cell communication in MMC differentiation. 29

30 The authors responsible for distribution of materials integral to the findings presented in this 31 article in accordance with the policy described in the Instructions for Authors 32 (www.plantcell.org) are: Xuemei Chen ([email protected]) and Yuan Qin 33 ([email protected]). 34

35

ABSTRACT 36

In the ovules of most sexually reproducing plants, one hypodermal cell differentiates 37

into a megaspore mother cell (MMC), which gives rise to the female germline. 38

Trans-acting small interfering RNAs known as tasiR-ARFs have been suggested to act 39

non-cell autonomously to prevent the formation of multiple MMCs by repressing 40

AUXIN RESPONSE FACTOR3 (ARF3) expression in Arabidopsis thaliana, but the 41

underlying mechanisms are unknown. Here, we examined tasiR-ARF-related 42

intercellular regulatory mechanisms. Expression analysis revealed that components of 43

the tasiR-ARF biogenesis pathway are restricted to distinct ovule cell types, thus 44

limiting tasiR-ARF production to the nucellar epidermis. We also provide data 45

suggesting tasiR-ARF movement along the medio-lateral axis into the hypodermal 46

Plant Cell Advance Publication. Published on July 23, 2020, doi:10.1105/tpc.20.00126

©2020 American Society of Plant Biologists. All Rights Reserved

mailto:[email protected]




2

cell and basipetally into the chalaza. Furthermore, we used cell type-specific 47

promoters to express ARF3m, which is resistant to tasiR-ARF regulation, in different 48

ovule cell layers. ARF3m expression in hypodermal cells surrounding the MMC, but 49

not in epidermal cells, led to a multiple-MMC phenotype, suggesting that tasiR-ARFs 50

repress ARF3 in these hypodermal cells to suppress ectopic MMC fate. RNA-seq 51

analyses in plants with hypodermally expressed ARF3m showed that ARF3 potentially 52

regulates MMC specification through phytohormone pathways. Our findings uncover 53

intricate spatial restriction of tasiR-ARF biogenesis, which together with tasiR-ARF 54

mobility, enables cell-cell communication in MMC differentiation. 55

56

INTRODUCTION 57

Germline specification is a crucial step in sexual reproduction. Plant reproduction is 58

initiated by the specification of sporocytes that form haploid spores through meiosis. 59

In most sexually reproducing plants, a single somatic, hypodermal cell in the ovule 60

differentiates into a megaspore mother cell (MMC), which gives rise to the female 61

germline (Grossniklaus, 2011; Yang et al., 2010). As the MMC is the first committed 62

cell of the female germline lineage, MMC specification and its regulation are of 63

critical importance. 64

Trans-acting small interfering RNAs (ta-siRNAs) are a class of endogenous 65

small interfering RNAs (Chen, 2009). The production of ta-siRNAs begins with the 66

transcription of TAS loci into long noncoding RNAs by Pol II (Peragine et al., 2004; 67

Vazquez et al., 2004; Yoshikawa et al., 2005). The TAS RNAs are exported by the 68

THO/TREX (‘transcription/export’) complex to the cytoplasm (Jauvion et al., 2010; 69

Yelina et al., 2010) where they are targeted for cleavage by ARGONAUTE (AGO) 70

guided by different microRNAs (miR173-AGO1 for TAS1 and TAS2, miR390-AGO7 71

for TAS3, and miR828-AGO1 for TAS4 in Arabidopsis thaliana) (Allen et al., 2005; 72

Luo et al., 2012; Montgomery et al., 2008a; Montgomery et al., 2008b; Peragine et al., 73

2004; Rajagopalan et al., 2006; Vazquez et al., 2004; Yoshikawa et al., 2005). The 74

cleaved RNA fragments are bound and stabilized by SUPPRESSOR OF GENE 75

SILENCING 3 (SGS3) and copied into double-stranded (ds) RNAs by 76

RNA-DEPENDENT RNA POLYMERASE 6 (Peragine et al., 2004; Vazquez et al., 77

2004; Yoshikawa et al., 2013; Yoshikawa et al., 2005). DICER-LIKE 4 then processes 78

the dsRNAs into phased 21 nt ta-siRNAs from one end of the dsRNAs defined by the 79

cleavage event (Gasciolli et al., 2005; Xie et al., 2005; Yoshikawa et al., 2005). The 80

3

ta-siRNAs are loaded into the effector AGO1 (Baumberger and Baulcombe, 2005). 81

The tasiR-ARFs from TAS3 loci are so named because they target several AUXIN 82

RESPONSE FACTOR (ARF) genes (Fahlgren et al., 2006; Garcia et al., 2006; Hunter 83

et al., 2006; Williams et al., 2005); they are conserved in angiosperms and play 84

critical roles in plant development (Allen et al., 2005; Dotto et al., 2014; Li et al., 85

2014; Marin et al., 2010; Nogueira et al., 2007; Ozerova et al., 2013; Wang et al., 86

2010; Xia et al., 2017). 87

Recent studies have identified a number of factors involved in MMC 88

differentiation in Arabidopsis (Cao et al., 2018; Li et al., 2017; Singh et al., 2017; Yao 89

et al., 2018). The preferential localization of some of these factors in different ovule 90

cell layers hints at intercellular signaling in MMC specification. We previously 91

showed that TRANSCRIPTION EXPORT 1 (TEX1), which encodes a protein in the 92

THO complex involved in the biogenesis of ta-siRNAs (Jauvion et al., 2010; Yelina et 93

al., 2010), is specifically expressed in ovule epidermal cells and represses ectopic 94

MMC fate by promoting the biogenesis of tasiR-ARFs. Accordingly, a mutation in 95

TEX1 leads to the formation of supernumerary MMCs from hypodermal cells (Su et 96

al., 2017). AGO9-dependent 24 nt siRNAs derived from transposable elements (TEs) 97

were proposed to be produced in epidermal cells and move to the hypodermal, 98

MMC-adjacent cells to prevent them from differentiating into MMCs 99

(Olmedo-Monfil et al., 2010). There may be a similar role for AGO104 during female 100

gametophyte formation in maize (Zea mays) (Singh et al., 2011). In Arabidopsis, the 101

transcription factor gene WRKY28 is expressed specifically in the hypodermal somatic 102

cells surrounding the MMC; mutations in WRKY28 also lead to multiple MMCs in 103

ovule primordia (Zhao et al., 2018). Arabidopsis MNEME (MEM), a putative RNA 104

helicase gene expressed specifically in the MMC, inhibits neighboring somatic cells 105

from acquiring MMC identity (Schmidt et al., 2011). These findings suggest that 106

intercellular signaling plays key roles in the restriction of the MMC fate to a single 107

cell. 108

Our previous studies found that TEX1 represses ectopic MMC fate by spatially 109

restricting the expression of ARF3 in Arabidopsis ovule primordia. Arabidopsis ovule 110

primordia are radially symmetrical structures with proximal-distal and medio-lateral 111

polarities. Three structural elements, the funiculus, chalaza and nucellus, can be 112

distinguished along the proximal-distal axis. In wild-type ovules, ARF3 is expressed 113

in the chalazal region, the central part of the ovule along the proximal-distal axis. 114

4

However, without proper repression by tasiR-ARFs, ARF3 is expressed both in the 115

central chalaza and the distal nucellus, including the cells adjacent to the MMC and 116

the outermost epidermal cells (Su et al., 2017). While the ectopic expression of ARF3 117

leads to the multiple-MMC phenotype, it is unclear how the tasiR-ARF pathway 118

operates in the ovule to achieve the repression of ARF3 in MMC-adjacent cells. 119

In the present study, we systematically interrogated the spatial patterns of 120

expression of genes required for tasiR-ARF biogenesis in ovule primordia. We found 121

that AGO7 expression is concentrated in the nucellar region along the proximal-distal 122

axis and SGS3 expression is restricted to the epidermis along the medio-lateral axis; 123

the restricted patterns together predict that tasiR-ARF biogenesis occurs in the 124

epidermal layer of the nucellus. We showed that an ago7 mutant exhibits a 125

multiple-MMC phenotype and that AGO7 expression in apical, epidermal cells in the 126

mutant was sufficient to rescue the multiple-MMC phenotype. In contrast, AGO1, the 127

protein effector of tasiR-ARFs, exhibited hypodermal cell enriched localization. 128

Furthermore, we found that the repression of ARF3 expression in hypodermal cells 129

surrounding the MMC is crucial to ensure the formation of a single MMC. These 130

findings support a mechanism in which tasiR-ARFs produced in the nucellar 131

epidermal cells repress ectopic MMC-like fate by targeting ARF3 transcripts in the 132

hypodermal cells adjacent to the MMC. 133

134

RESULTS 135

TAS3 is transcribed ubiquitously in Arabidopsis ovules 136

To determine the sites of tasiR-ARF biogenesis in ovule primordia, we first 137

investigated the expression patterns of TAS3 loci, which are transcribed into 138

precursors to tasiR-ARFs, using GUS reporter constructs driven by the TAS3A and 139

TAS3B promoters. The constructs were introduced into wild type Arabidopsis to 140

generate pTAS3A:GUS and pTAS3B:GUS stable transformants. Note that TAS3C is 141

not expressed at detectable levels in ovules (Su et al., 2017). 142

GUS histochemical analysis was performed in six to ten independent T1 143

transgenic lines for each construct, and typical examples are shown. GUS staining 144

was not detected in control wild type ovules (Supplemental Figure 1A-B). Strong 145

GUS activity was detected in ovules of pTAS3A:GUS and pTAS3B:GUS 146

transformants, and the results indicated that TAS3A and TAS3B have similar, 147

ubiquitous expression patterns during ovule development. At the MMC stage, GUS 148

5

staining was detected throughout the ovule and in the transmitting tract (Supplemental 149

Figure 1D and 1G), and similar patterns were observed in meiosis stage ovules 150

(Supplemental Figure 1E and 1H). As the ovules matured, however, GUS activity 151

became weaker (Supplemental Figure 1F and 1I). 152

153

MIR390 and AGO7 are involved in MMC specification 154

Long noncoding RNAs from the TAS3 loci are targeted by the miR390-AGO7 155

complex to trigger tasiR-ARF production (Allen et al., 2005; Endo et al., 2013; 156

Montgomery et al., 2008a). TAS3A and TAS3B have been shown to regulate MMC 157

specification – the tas3a and tas3b mutants exhibit a multiple-MMC phenotype in 158

approximately 20% of ovules (Su et al., 2017). However, it is not known whether 159

MIR390 or AGO7 has similar functions in MMC differentiation. We tested this using 160

mir390 and ago7 mutants. There are two MIR390 genes and the mir390 52b2 mutant 161

(hereafter referred to as mir390) in MIR390A has reduced levels of miR390 and 162

tasiR-ARFs (Cuperus et al., 2010). Using differential interference contrast (DIC) 163

observation, the MMC phenotype was analyzed as the frequency of pre-meiotic 164

ovules with more than one enlarged cell. In mir390, ovule primordia containing more 165

than one MMC-like cells occurred at a frequency of 20.2% (Figure 1B), which is 166

higher than that in wild type (3.2%, n=94) (Figure 1A, 1M) and similar to those of 167

tas3a and tas3b (Su et al., 2017). For AGO7, we examined two mutants, ago7-9 168

(SALK_080533) and zip-2 (Hunter et al., 2006), and the frequencies of ovule 169

primordia with multiple MMC-like cells in these mutants were 24.6% and 22.3%, 170

respectively, which were significantly higher than that in WT (Figure 1C,1D and 1M). 171

The results above indicated that loss-of-function in MIR390 and AGO7 phenocopy 172

tas3 in having supernumerary MMC-like cells. 173

To determine whether the supernumerary MMC-like cells in mir390 and ago7 174

had MMC characteristics, we introduced pKNU:KNU-Venus into mir390 and ago7 by 175

crossing. KNU-Venus was detected in the single MMC of wild type ovule primordia 176

(Figure 1E). In mir390, KNU-Venus was observed in more than one cell in 19.7% of 177

ovules. In zip-2 and ago7-9, KNU-Venus was observed in multiple cells in 16.3% and 178

19.1% of ovules, respectively (Figure 1F-1H and 1N). The nuclear localization of 179

AGO9 serves as another MMC marker (Rodriguez-Leal et al., 2015) (Figure 1I). 180

Whole-mount immunolocalization of AGO9 in these mutants showed that the 181

frequency of pre-meiotic ovules with AGO9 nuclear localization in multiple 182

6

MMC-like cells was higher in mir390, zip-2 and ago7-9 than in wild type (Figure 183

1I-1L and lO). Together, the KNU-Venus expression data and the AGO9 localization 184

analysis indicated that the multiple, enlarged MMC-like cells in pre-meiotic mir390 185

and ago7 ovules acquired MMC characteristics. 186

To examine whether one or more of the MMC-like cells present in mir390, 187

ago7-9 and zip-2 could undergo meiosis, we performed immunolocalization with 188

antibodies against DMC1, which is specifically expressed in the megasporocyte 189

undergoing meiosis in wild type ovules (Figure 2A) (Imran Siddiqi, 2000; Klimyuk 190

and Jones, 1997). In mir390, ago7-9 and zip-2 ovules, DMC1 signal was only 191

detected in one MMC and not in the other MMC-like cells (Figure 2B-2D). 192

In addition to DMC1, we also analyzed callose deposition, another reliable 193

marker for MMCs undergoing meiosis (Mary C. Webb, 1990). In wild type ovules 194

during meiosis, callose was deposited in the transverse walls between the functional 195

megaspore and its degenerated sister cells (Figure 2E and 2I). In mir390 post-meiotic 196

ovules, callose was detected in the walls between the single functional megaspore and 197

the degenerated neighboring cells, but it was not detected at the additional, 198

abnormally enlarged cells (Figure 2F and 2J). Similar callose staining results were 199

obtained in the zip-2 (Figure 2G and 2K) and ago7-9 (Figure 2H and 2L) mutants. 200

Based on these findings, we concluded that although multiple MMC-like cells 201

differentiated in mir390, ago7-9 and zip-2 ovules, only one underwent meiosis. Thus, 202

the enlarged cells only acquired partial MMC identity. Nevertheless, both MIR390 and 203

AGO7 are required to suppress ectopic MMC specification. 204

205

miR390 is highly concentrated in the nucellar epidermis 206

Having shown that MIR390A is required for the spatial precision in MMC 207

specification, we next studied its expression in ovules using a reporter gene encoding 208

beta glucuronidase (GUS). In pMIR390A:GUS transformants, strong GUS activity 209

was detected throughout MMC stage ovules (Figure 3A), indicating that MIR390A 210

was transcribed ubiquitously. We then examined miR390 accumulation in ovules by 211

in situ hybridization with an LNA-modified probe complementary to miR390. In 212

longitudinal sections of ovules, miR390 was present throughout the chalazal region 213

but was concentrated in the epidermis in the nucellar region (Figure 3B). These 214

patterns were further confirmed with cross sections of ovules: signals were nearly 215

absent in the center of the ovule at the base of the nucellar region but was throughout 216

7

the medial-lateral axis in the chalazal region (Figure 3D, E). No signals were detected 217

in wild type ovules incubated without the probe (Figure 3C, F). Thus, despite 218

ubiquitous transcription of MIR390A in the ovule, the mature miRNA was at low 219

levels in the MMC and the surrounding cells. This implicated an unknown 220

posttranscriptional mechanism that impacts miR390 accumulation. 221

222

AGO7 is expressed in the distal nucellus of ovule 223

To determine the expression patterns of AGO7 in ovules, we generated the promoter 224

reporter line pAGO7:GUS as well as the protein reporter line pAGO7:GFP-AGO7. 225

Expression analyses were performed with five T1 transgenic plants. In 226

pAGO7:GFP-AGO7 transgenic lines, GFP signal was concentrated in the nucellus, in 227

which cells adjacent to the MMC had particularly strong signals and epidermal cells 228

and the MMC had weaker signals (Figure 3G and 3H). Similar but more strikingly 229

restrictive expression patterns were observed in the promoter reporter lines 230

pAGO7:GUS (Figure 3I). 231

232

SGS3 expression is restricted to epidermal cells 233

Following miR390-AGO7-mediated cleavage of TAS3 transcripts, the cleavage 234

fragments are stabilized by SGS3 in tasiR-ARF biogenesis (Yoshikawa et al., 2013; 235

Yoshikawa et al., 2005). It was previously reported that mutations in SGS3 induce 236

multiple MMCs in Arabidopsis ovule primordia (Olmedo-Monfil et al., 2010). We 237

analyzed SGS3 expression in ovule primordia using a fusion to green fluorescent 238

protein (GFP) or GUS (pSGS3:SGS3-GFP and pSGS3:SGS3-GUS). In 239

pSGS3:SGS3-GFP transgenic lines at the early megasporogenesis stage, SGS3-GFP 240

accumulated specifically in the ovule epidermal cell layer (Figure 3J-L). Similar 241

results were obtained in ovules at the later megasporogenesis stage (Supplemental 242

Figure 2A-2C). The SGS3-GUS expression pattern was also consistent with the 243

SGS3-GFP results (Supplemental Figure 2D). The spatial pattern of SGS3 expression 244

resembled that of TEX1 (Su et al., 2017), which is involved in the nuclear export of 245

TAS3 RNAs in tasiR-ARF biogenesis (Yelina et al., 2010). Thus, the preferential 246

expression of SGS3 and TEX1 in the epidermal cell layer of ovule primordia may 247

result in the spatially restricted production of tasiR-ARFs in the ovule. 248

249

Apical epidermal expression of AGO7 is sufficient to rescue the MMC phenotype 250

8

of ago7 251

The results above show that AGO7 expression is mainly in the distal nucellus (Figure 252

3D-3F) and SGS3 expression is restricted to the epidermis (Figure 3G-3I). As both 253

AGO7 and SGS3 are required for tasiR-ARF biogenesis, the restricted spatial 254

expression patterns of the two genes probably limit tasiR-ARF biogenesis to the 255

epidermis of the nucellus. If this were the case, we would expect that AGO7 256

expression in the epidermis should be sufficient for MMC specification. We 257

expressed AGO7 with cell layer-specific promoters in the ago7-9 background to 258

determine where AGO7 expression is necessary to rescue the multiple-MMC 259

phenotype of ago7-9. 260

In Arabidopsis, the transcription factor gene WRKY28 is exclusively expressed in 261

the hypodermal somatic cells surrounding the MMC (Zhao et al., 2018), so we used 262

the WRKY28 promoter to specifically express AGO7 in these hypodermal cells 263

(pWRKY28:GFP-AGO7). Consistent with the WRKY28 expression pattern, GFP 264

signal was specifically detected in the hypodermal cells surrounding the MMC in 265

pWRKY28:GFP-AGO7 ago7-9 transgenic plants (Figure 4A and 4B). The frequency 266

of pre-meiotic ovules with more than one MMC-like cells in pWRKY28:GFP-AGO7 267

ago7-9 lines was 22.5% (n=182) (Figure 4C), comparable to that in ago7 (Figure 1C 268

and 1D). Thus, expression of AGO7 in the hypodermal cells surrounding the MMC is 269

unable to rescue the MMC specification defects in ago7-9. 270

SPOROCYTELESS (SPL) is required for the differentiation of the megasporocyte 271

and microsporocytes, and it is expressed specifically in the apical epidermal cells at 272

the MMC stage in ovules (Wei et al., 2015; Yang et al., 1999). In pSPL:GFP-AGO7 273

ago7-9 transgenic plants, the GFP-AGO7 fusion protein was located in the outermost 274

cells of ovule nucellus (Figure 4D and 4E). Ovules with more than one enlarged cell 275

were detected in these transgenic lines at a low frequency of 4.6% (Figure 4F), 276

comparable to that in wild type, indicating that AGO7 expression in the apical 277

epidermal cells could rescue the multiple-MMC phenotype of ago7-9. These results 278

also suggested that the production of tasiR-ARFs in apical epidermal cells is critical 279

for MMC differentiation in Arabidopsis. 280

281

TasiR-ARFs repress ARF3 expression in cells adjacent to the MMC to prevent 282

ectopic MMC differentiation 283

TasiR-ARFs need to be produced in the epidermal cells of ovule nucellus for correct 284

9

MMC specification. In which cells do tasiR-ARFs repress ARF3 expression? To 285

determine the sites of tasiR-ARF function, we analyzed the expression patterns of 286

ARF3 in ovule primordia with or without proper regulation by tasiR-ARFs by 287

whole-mount in situ hybridization. We compared ARF3 expression between wild type 288

and the rdr6-11 mutant, which has greatly reduced levels of tasiR-ARFs (Peragine et 289

al., 2004; Su et al., 2017; Yoshikawa et al., 2005). At the early MMC stage in wild 290

type, ARF3 mRNA was detected at the basal end of the chalaza along the 291

proximal-distal axis (Figure 5A). In rdr6-11, ARF3 expression expanded to the apical 292

side of the chalazal region, and weak signals were also detected in the nucellus 293

(Figure 5B). Similar results were observed in late MMC stage ovules (Figure 5D and 294

5E). No signals were detected in samples with the ARF3 sense probe or without probe 295

(Figure 5C and 5F). The in situ hybridization results were consistent with the 296

ARF3-GFP and ARF3m-GFP patterns (resistant to regulation by the tasiR-ARFs) or 297

ARF3-GFP patterns in tex1 (defective in the production of tasiR-ARFs) reported 298

previously (Su et al., 2017), with ARF3-GFP mainly in the central chalaza and 299

ARF3m-GFP, as well as ARF3-GFP in tex1, in both the central chalaza and distal 300

nucellus. Thus, the activities of tasiR-ARFs exhibited apical polarity in the ovule, 301

with the nucellus and the apical portion of the chalaza being the sites of action. 302

The above analyses suggest that tasiR-ARFs repress ARF3 expression in a broad 303

apical domain in ovules, but it is unknown in which cells the repression is necessary 304

for correct MMC specification. To determine whether the development of multiple 305

MMC-like cells was attributable to the expression of ARF3m in a specific cell type, 306

we used specific promoters to express ARF3m in different ovule cell layers in wild 307

type plants. 308

In pSPL:ARF3m-mCitrine transgenic plants, the ARF3m-mCitrine fusion protein 309

was located in the apical epidermal cells of ovule primordia (Figure 6A-6C). In 310

pSPL:ARF3m-mCitrine transgenic lines, the frequency of ovules with >1 MMC-like 311

cells was 5.3% (Figure 6D), comparable to that in wild type, indicating that ARF3m 312

expression in the outermost epidermal cells can not result in multiple enlarged cells in 313

ovule primordia. In pWRKY28:ARF3m-mCitrine transgenic lines, mCitrine signal was 314

specifically detected in the hypodermal cells surrounding the MMC (Figure 6E-6G). 315

DIC observation of the MMC phenotype in pWRKY28:ARF3m-mCitrine indicated 316

that pre-meiotic ovules with more than one enlarged cell occurred at a frequency of 317

23.5% (n=196) (Figure 6H), much higher than that in wild type and similar to that in 318

10

pARF3:ARF3m-GFP (Su et al., 2017), suggesting that ARF3m expression in the 319

hypodermal cells is sufficient to cause the formation of enlarged cells in ovule 320

primordia. For both pWRKY28:ARF3m-mCitrine and pSPL:ARF3m-mCitrine, 321

RT-qPCR analysis confirmed an increase in ARF3 transcript levels compared to wild 322

type (Supplemental Figure 3). 323

To investigate whether enlarged cells in pWRKY28:ARF3m-mCitrine had MMC 324

characteristics, we introduced pKNU:nlsGUS, a marker of MMC identity (Cao et al., 325

2018; Payne et al., 2004) into the transgenic lines by crossing. In wild type ovule 326

primordia, GUS was detected in the single MMC (Figure 6I). In 327

pWRKY28:ARF3m-mCitrine, GUS was observed in two or even three MMC-like cells 328

at a frequency of 20.2%, which was higher than that in wild type (Figure 6J and 6K). 329

We also performed whole-mount immunolocalization with anti-AGO9 antibodies. In 330

wild type, AGO9 nuclear localization was detected only in the single MMC 331

(Olmedo-Monfil et al., 2010). In pWRKY28:ARF3m-mCitrine, 21.4% of the analyzed 332

ovule primordia had AGO9 nuclear localization in more than one cell (Figure 6L), 333

indicating that the enlarged cells in these transgenic ovule primordia had acquired 334

MMC characteristics. Taken together, these results showed that ARF3m expression in 335

the hypodermal cells, but not in the outermost epidermal cells, resulted in multiple 336

MMC-like cells in ovule primordia. 337

The tasiR-ARFs are recruited into an AGO1-containing effector complex to 338

repress ARF3 expression (Baumberger and Baulcombe, 2005). In light of the role of 339

AGO1 in mediating the functions of tasiR-ARFs, we investigated the spatial patterns 340

of AGO1 expression in Arabidopsis ovule primordia using pAGO1:YFP-AGO1. At the 341

early megasporogenesis stage, YFP-AGO1 signal was detected in the distal nucellus 342

with preferential expression in hypodermal cells (Supplemental Figure 4A-4C). This 343

localization pattern was consistent with the finding that the suppression of ARF3 by 344

tasiR-ARFs occurred in the hypodermal cells surrounding the MMC. 345

346

Genes regulated by ARF3 347

To broaden our investigation of how ARF3m expression in ovule hypodermal 348

cells led to the multiple-MMC phenotype, we performed RNA-seq using gynoecia at 349

the MMC stage from WT and pWRKY28:ARF3m-mCitrine transgenic plants to 350

identify potential downstream genes of ARF3. Two replicates were performed and 351

reproducibility was high (Supplemental Figure 5). The analysis identified 447 352

11

differentially expressed genes (DEGs) (Supplemental Data Set 1), which we 353

considered as candidate target genes regulated by ARF3. We compared the publicly 354

available arf3 mutant gynoecium RNA-seq data (Simonini et al., 2017; Zheng et al., 355

2018) with our pWRKY28:ARF3m RNA-seq data and found only 23 genes in common 356

(Supplemental Data Set 2) between the 447 DEGs in pWRKY28:ARF3m vs. WT and 357

the 738 DEGs in arf3 vs. WT, indicating that downstream genes regulated by ARF3 358

during MMC differentiation and gynoecium development were largely different. 359

GO analysis of these 447 DEGs revealed an enrichment of the terms “cellular 360

process” and “metabolic process” (Supplemental Figure 6). KEGG pathway 361

enrichment analysis indicated that genes associated with metabolism and plant 362

hormone signal transduction pathways were enriched among the 447 DEGs 363

(Supplemental Figure 7). It has been reported that during the development of ovule 364

primordia, auxin and cytokinin have polarized distributions, with auxin at the distal 365

end and cytokinin at the proximal end (Shirley et al., 2019). Taken together, these 366

results suggest that phytohormones may be involved in MMC differentiation. 367

However, the mechanisms through which hormones exert their functions during this 368

process are still unclear. 369

370

DISCUSSION 371

In this study, we revealed distinct spatial patterns of expression of key components in 372

tasiR-ARF biogenesis in the ovule. From these patterns, it can be deduced that 373

tasiR-ARF production is restricted to the nucellar epidermis. However, the activities 374

of tasiR-ARFs were detected in broader domains, consistent with the mobility of 375

tasiR-ARFs. Furthermore, we found that the expression of ARF3m, which is resistant 376

to tasiR-ARF regulation, in the hypodermal cells surrounding the MMC resulted in a 377

multiple-MMC phenotype, suggesting that tasiR-ARFs repress ARF3 expression in 378

the apical, hypodermal cells to prevent them from adopting an MMC-like fate. The 379

study uncovered extensive coordination between cell layers during MMC 380

specification. 381

382

TasiR-ARF mobility in Arabidopsis ovule primordia 383

TAS3 genes are expressed nearly ubiquitously in ovules, but SGS3 and AGO7 have 384

restricted expression patterns in the radial and proximal-distal axes, respectively. 385

SGS3 is only expressed in the epidermis and AGO7 expression is mainly in the distal 386

12

nucellus. As both SGS3 and AGO7 are required for tasiR-ARF biogenesis, the 387

restricted spatial patterns of the two genes probably limit tasiR-ARF biogenesis to the 388

epidermis of the nucellus. In fact, we demonstrated that expression of AGO7 in the 389

distal-most epidermal cells was sufficient to rescue the multiple-MMC phenotype of 390

an ago7 mutant. The spatial pattern of miR390 accumulation is intriguing: the 391

miRNA is concentrated in the epidermis along the radial axis despite ubiquitous 392

promoter activity. Thus, miR390 is largely excluded from the MMC and its 393

surrounding cells. While it is unknown how this pattern is established, the 394

predominant localization of miR390 in epidermal cells may serve as another piece of 395

evidence for tasiR-ARF biogenesis in the nucellar epidermis. 396

TasiR-ARFs guide ARF3 RNA cleavage through their incorporation into an 397

AGO1-containing effector complex (Baumberger and Baulcombe, 2005). The present 398

observation that AGO1 accumulates in the hypodermal cells surrounding the MMC 399

suggests that the cleavage of ARF3 mRNA by tasiR-ARFs occurs in these cells. This 400

is consistent with the finding that repression of ARF3 expression in hypodermal cells 401

is crucial for the suppression of the MMC-like fate. 402

Previous studies have provided strong evidence that tasiR-ARFs move across 403

cell layers during leaf development. While TAS3 is transcribed in the epidermal layer 404

to produce the tasiR-ARF precursors, tasiR-ARF abundance follows a gradient 405

emanating from the epidermal layer of leaf primordia (Chitwood et al., 2009). Our 406

study indicates that there may be similar movement of tasiR-ARFs during female 407

gametophyte formation in Arabidopsis. Although our efforts at in situ hybridization to 408

determine the localization of tasiR-ARFs in ovules failed, the expression patterns of 409

different proteins required for tasiR-ARF biogenesis or action suggest the following 410

model. Along the radial axis, tasiR-ARFs are produced in ovule epidermal cells but 411

function in hypodermal cells (Figure 7). Along the proximal-distal axis, tasiR-ARFs 412

are produced in the nucellus but moves into the distal chalaza to repress ARF3 413

expression (Figure 7). 414

415

Cellular competence to acquire MMC identity in the hypodermal cell layer 416

In ovules of most sexually reproducing flowering plants, female gametogenesis is 417

initiated from a single gametic cell derived from the MMC (Walbot and Evans, 2003). 418

Because some mutants and certain sexual species develop more than one MMC 419

(Bicknell and Koltunow, 2004; Cao et al., 2018; Olmedo-Monfil et al., 2010; Su et al., 420

13

2017; Yao et al., 2018; Zhao et al., 2018), and many species are able to form gametes 421

without meiosis (through apomixis) (Bicknell and Koltunow, 2004), it has been 422

proposed that somatic cells in the ovule have the competence to obtain MMC identity. 423

In Arabidopsis and most angiosperms, the position of the single germline 424

precursor is precisely controlled. Early in ovule development, two layers (L) can be 425

distinguished in the nucellus, the L1 epidermal layer and the L2 hypodermal layer. In 426

Arabidopsis, the distal-most L2 cell becomes the germline MMC. Previous studies 427

have demonstrated that in addition to the distal-most cell, other hypodermal cells have 428

the potential to become MMCs. Monfil et al. propose that TE-derived 24 nt siRNAs 429

produced in epidermal (L1) cells move to the hypodermal nucellar cells to prevent 430

them from differentiating into MMCs (Olmedo-Monfil et al., 2010). Arabidopsis KLU 431

acts through the chromatin remodeling complex SWR1 to promote WRKY28 432

expression specifically in the hypodermal somatic cells surrounding the MMC, and 433

WRKY28 is required to prevent these cells from acquiring MMC-like characteristics 434

(Zhao et al., 2018). In the present study, we found that tasiR-ARFs repress ARF3 435

expression in the hypodermal cells to prevent these cells from gaining MMC-like 436

characteristics. To our knowledge, no genes have been reported to be involved in cell 437

identity transitions from epidermal cells to MMC. In contrast, the derivation of 438

MMC-like cells from hypodermal cells occurs in a number of mutants. This suggests439

that hypodermal somatic cells have more potential to acquire MMC identity. 440

Our data showed that the expression of ARF3 in hypodermal cells adjacent to the 441

MMC promotes the hypodermal cells to gain MMC-like identity, suggesting it is a 442

positive factor for MMC formation. However, its positive effect is limited. In the 443

present study, the expression of ARF3 in the outermost epidermal cells cannot cause 444

the cell fate change from epidermal cell to MMC, suggesting that the expression of 445

ARF3 is not sufficient for MMC formation. Moreover, in the MMC itself, ARF3 is not 446

expressed, suggesting that it is not necessary for true MMC fate. These findings 447

implicate the presence of complex regulatory networks in different ovule cell layers 448

and suggest that ARF3 needs to be repressed in the hypodermal cells surrounding the 449

MMC to prevent these cells from adopting an MMC-like fate. 450

In summary, this and prior studies show that there are complex regulatory 451

mechanisms that prevent hypodermal cells from gaining MMC identity during ovule 452

development in Arabidopsis. Furthermore, other regulatory factors, such as SPL and 453

WUS, which promote the formation of the actual MMC, have also been revealed. 454

14

Mutants of these genes fail to form the actual MMC (Gross-Hardt et al., 2002; 455

Schiefthaler et al., 1999; Yang et al., 1999). This suggests that dual regulatory 456

mechanisms acting on the MMC and the surrounding cells ensure precise MMC 457

differentiation. 458

459

MATERIALS AND METHODS 460

Plant materials and growth conditions 461

Wild-type Arabidopsis thaliana Columbia-0 (Col-0), mir390 (mir390 52b2) (Cuperus 462

et al., 2010), ago7-9 (SALK_080533), MIR390:GUS (CS66476) and zip-2 (CS24282) 463

(Hunter et al., 2006) were grown under 16 h light (50 μmol m-2

s-1

, white light)/8 h464

dark conditions at 22°C. 465

466

Plasmid construction 467

For the pTAS3A:GUS and pTAS3B:GUS plasmids, a 2150 bp sequence upstream of 468

TAS3A and a 1463 bp sequence upstream of TAS3B were amplified from wild type 469

genomic DNA using primers listed in Supplemental Table 1. The PCR fragments were 470

cloned into the pENTR/D-TOPO (Invitrogen) vector and verified by sequencing. The 471

fragments were subsequently recombined into the destination vector pMDC162 472

(Curtis and Grossniklaus, 2003) using LR Clonase II (Invitrogen). 473

For pSPL:ARF3m-mCitrine, a 2704 bp sequence upstream of SPL was amplified 474

as the promoter pSPL from wild type genomic DNA using the primers pSPL-F and 475

pSPL-R (Supplemental Table 1). The ARF3m fragment was amplified from the 476

pARF3:ARF3m-GFP plasmid (Liu et al., 2014) using primers ARF3m-F and 477

ARF3m-R (Supplemental Table 1). The pSPL:ARF3m fragment was generated by 478

PCR using a mixture of pSPL and ARF3m as the template and cloned into the 479

pENTR/D-TOPO (Invitrogen) vector. The presence of the tasiR-ARF-resistant 480

mutation in pSPL-ARF3m was verified by sequencing. Finally, recombination into the 481

destination vector pGWB5012 (Nakagawa et al., 2007) was performed using LR 482

Clonase II to generate pSPL:ARF3m-mCitrine. For pWRKY28:ARF3m-mCitrine, a 483

2097 bp sequence upstream of WRKY28 was amplified using the primers pWRKY28-F 484

and pWRKY28-R (Supplemental Table 1). The subsequent steps to construct 485

pWRKY28:ARF3m-mCitrine were as described above for pSPL:ARF3m-mCitrine. 486

For pAGO7:GUS, a 2081 bp sequence upstream of AGO7 was amplified as the 487

promoter pAGO7 using primers pAGO7-F and pAGO7-R (Supplemental Table 1). 488

https://www.arabidopsis.org/servlets/TairObject?type=germplasm&id=1007965150

15

The PCR fragment was cloned into pENTR/D-TOPO (Invitrogen) and verified by 489

DNA sequencing, followed by recombination into the destination vector pGWB533 490

(Nakagawa et al., 2007) using LR Clonase II. 491

To generate the pAGO7:GFP-AGO7, pSPL:GFP-AGO7 and 492

pWRKY28:GFP-AGO7 constructs, the pAGO7, pSPL and pWRKY28 promoters were 493

amplified from Arabidopsis genomic DNA as described above, and the GFP fragment 494

was amplified from pGWB504 (Nakagawa et al., 2007). The AGO7 sequence was 495

amplified by PCR using Arabidopsis cDNA as the template using primers AGO7cds-F 496

and AGO7cds-R (Supplemental Table 1). pAGO7:GFP-AGO7, pSPL:GFP-AGO7 and 497

pWRKY28:GFP-AGO7 were cloned into pENTR/D-TOPO by infusion (Invitrogen), 498

followed by recombination into the destination vector pGWB501 (Nakagawa et al., 499

2007) using LR Clonase II. 500

To construct pSGS3:SGS3-GFP and pSGS3:SGS3-GUS, a genomic fragment 501

encompassing 2148 bp upstream of the start codon of SGS3 and a 2251 bp SGS3 502

coding region without the stop codon was amplified with primers pSGS3-F and 503

SGS3-R. The PCR product was cloned into pENTR/D-TOPO and verified by 504

sequencing. The fragment was recombined by LR reaction into the destination vectors 505

pGWB504 (Nakagawa et al., 2007) and pGWB533 (Nakagawa et al., 2007) to obtain 506

pSGS3:SGS3-GFP and pSGS3:SGS3-GUS, respectively. 507

508

Preparation of ovules for confocal laser scanning microscopy 509

Ovules were dissected, stained in 40% glycerol with 5 mM FM4-64 for 5 min then 510

analyzed using a Leica TCS SP8 microscope. For the expression pattern analysis of 511

the transgenic lines, more than five independent transgenic lines were observed and 512

confirmed to have similar patterns. 513

514

Quantitative real-time PCR 515

To measure the relative transcript levels of selected genes, real-time quantitative PCR 516

(qPCR) was performed with specific primers (Supplemental Table 1) using the 517

Bio-Rad real-time PCR system and SYBR Premix Ex Taq II (TaKaRa) according to 518

the manufacturer’s instructions. 519

520

DIC observation of ovule structures 521

Ovules from wild type, mutant and transgenic plants were dissected from the pistils of 522

16

stage 8–13 flowers (Robinson-Beers et al., 1992) in a drop of chloral hydrate solution 523

(chloral hydrate:H2O:glycerol=8:2:1). An Olympus (BX63) microscope with DIC 524

optics was used to obtain images of the cleared ovules under a 40× objective. 525

526

GUS staining 527

Inflorescence samples were fixed in pre-chilled 80% acetone for 20 min then washed 528

with distilled water. After brief vacuum infiltration, the inflorescences were incubated 529

in β-glucuronidase (GUS) staining buffer (Oh et al., 2010) overnight at 37°C. The 530

pistils were dissected from the inflorescences then observed under an Olympus (BX63) 531

microscope. 532

533

Whole-mount immunolocalization in ovules 534

Ovules were dissected from 30–40 pistils of stage 9–11 flowers (Robinson-Beers et al., 535

1992) then fixed and processed according to a previously published protocol 536

(Escobar-Guzman et al., 2015). The AGO9 primary antibody (Agrisera, AS10673) 537

and DMC1 primary antibody (ABclonal Technology) were used at a dilution of 1:100. 538

The Alexa Fluor 488 secondary antibody (Molecular Probes) was used at a dilution of 539

1:300. The samples were stained with propidium iodide (PI, 500 mg/mL) before 540

mounting, and images were captured using a confocal microscope (Leica TCS SP8). 541

For PI detection, the excitation and emission wavelengths were 568 nm and 575–615 542

nm, respectively. For Alexa Fluor 488 detection, the excitation and emission 543

wavelengths were 488 nm and 500–550 nm, respectively. The laser intensity and gain 544

were set to the same levels for all analyzed genotypes. 545

546

ARF3 whole-mount in situ hybridization in ovules 547

Ovules with placenta were dissected from pistils of stage 9–11 flowers then fixed and 548

processed as previously described (Zhao et al., 2018). For the ARF3 probe, a 209 bp 549

cDNA fragment was amplified by PCR with primers ARF3-IS-F and ARF3-IS-F 550

(Supplemental Table 1) and cloned into the pTA2 vector (Toyobo). Generation of 551

digoxigenin-labeled probe was performed as described (Sieber et al., 2004). Ovule 552

whole-mount in situ hybridization was performed according to Hejátko et al (Hejatko 553

et al., 2006). 554

555

miR390 in situ hybridization for paraffin-embedded ovules 556

17

The sequence of the LNA probe to detect mature miR390 is 557

GGCGCUA+UC+CC+UCC+UGAGCUU (GenScript), in which “+” represents the 558

LNA position. Developing floral buds at the MMC stage were fixed and embedded in 559

Paraplast Plus embedding medium, cut into 6-μm sections, and then hybridized to the 560

probe as previously described (Kidner and Timmermans, 2006). 561

562

Callose detection 563

Aniline blue staining and callose detection in the megaspores were performed as 564

previously described (Imran Siddiqi, 2000) with minor modifications, including 565

fixation of inflorescences in FAA for 16 h and incubation in 0.1% aniline blue in 50 566

mM phosphate buffer (pH 11) for 8-12 h. The stained pistils were mounted in 30% 567

glycerol and observed with a BX63 microscope (Olympus) at an excitation 568

wavelength of 365 nm and using an emission long-pass filter of 420 nm. 569

570

RNA-seq and data analysis 571

RNA was extracted from gynoecia at the MMC stage (Robinson-Beers et al., 1992) 572

from wild type and pWRKY28:ARF3m-mCitrine plants using the Qiagen RNeasy kit. 573

Illumina sequencing was performed using 1 μg RNA per sample and two independent 574

biological replicates per genotype. The raw reads were filtered by removing the 575

adapter sequences and low-quality reads. Using the Arabidopsis thaliana TAIR10 576

assembly as the reference genome, the clean reads were aligned using STAR v2.5.0, 577

and the alignment results were processed using the SourceForge Subread package 578

featureCount v1.5.0 for RNA quantification. EdgeR v3.12.0 was used to identify 579

differentially expressed genes (fold change ≥ 2, false discovery rate ≤ 0.05) between 580

samples. 581

582

GO and pathway enrichment analysis 583

Gene ontology enrichment analysis for differentially expressed gene sets was 584

performed using TBtools (Chen et al., 2018). The P-value for enrichment was 585

calculated for each represented GO term and corrected via the Benjamini Hoschberg 586

error correction method (Ferreira and Nyangoma, 2008). The GO terms exhibiting a 587

corrected P-value of ≤0.05 were considered to be significantly enriched. Further, 588

pathway enrichment analysis of different sets of genes was also performed using 589

18

TBtools. 590

591

Accession Numbers 592

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

GenBank/EMBL databases under the following accession numbers: TAS3A 594

(AT3G17185), TAS3B (AT5G49615), ARF3 (AT2G33860), WRKY28(AT4G18170), 595

SPL (AT4G27330), AGO7(AT1G69440), MIR390(AT2G38325), SGS3(AT5G23570), 596

AGO1 (AT1G48410). The RNA-seq data were deposited in the European Nucleotide 597

Archive under accession number PRJEB38604. 598

599

SUPPLEMENTAL DATA 600

Supplemental Figure 1. pTAS3:GUS expression in Arabidopsis ovules. 601

Supplemental Figure 2. SGS3 expression in later MMC stage Arabidopsis ovules. 602

Supplemental Figure 3. ARF3 transcript levels in WT and the transgenic lines 603

pSPL:ARF3m-mCitrine and pWRKY28:ARF3m-mCitrine. 604

Supplemental Figure 4. AGO1 expression in Arabidopsis MMC-stage ovules. 605

Supplemental Figure 5. The consistency of RNA-seq replicates for WT and 606

pWRKY28:ARF3m-mCitrine. 607

Supplemental Figure 6. GO analysis of DEGs in pWRKY28:ARF3m-mCitrine 608

compared to WT. 609

Supplemental Figure 7. KEGG pathway enrichment analysis of DEGs in 610

pWRKY28:ARF3m-mCitrine compared to WT. 611

Supplemental Table 1. Primers and probes used in this study. 612

Supplemental Data Set 1. The DEGs of pWRKY28:ARF3m-mCitrine compared to 613

wild-type. 614

Supplemental Data Set 2. The DEGs in common between pWRKY28: ARF3m vs. WT 615

and arf3 vs. WT. 616

617

AUTHOR CONTRIBUTIONS 618

Z.S., Y.Q., and X.C. conceived the project; Z.S., N.W., Z.H. designed and performed619

the experiments; B.L., D.L., Y.L., H.C. analyzed the data; Z.S., Y.Q., and X.C. wrote 620

the manuscript. 621

622

https://www.arabidopsis.org/servlets/TairObject?id=1000529609&type=locus

19

ACKNOWLEDGEMENTS 623

We are grateful to Dr. Xigang Liu (Institute of Genetics and Developmental Biology, 624

Chinese Academy of Sciences [CAS]) for the kind gift of ARF3:ARF3m-GFP plasmid 625

and to Dr. H. Wang (Dept. of Biochemistry, University of Saskatchewan) for sharing 626

the pKNU:nlsGUS plasmid. This work was supported by grants from the Guangdong 627

Innovation Research Team Fund (2014ZT05S078), an NSFC grant (31700278) and 628

a China Postdoctoral Science Foundation Grant (2018T110888) to Z.S.; NSFC grants 629

(31970333; 31761130074), a Guangxi Distinguished Experts 630

Fellowship and a Newton Advanced Fellowship (NA160391) to Y.Q; an NIH grant 631

GM129373 to X.C. 632

633

Figure legends 634

Figure 1. MMC-like cells form in mir390 and ago7 ovules. 635

(A) WT ovule with one MMC. (B-D) Two MMC-like cells in a mir390 ovule (B), a636

zip-2 ovule (C) and an ago7-9 ovule (D). (E) Signal corresponding to pKNU: 637

KNU-Venus was detected in one cell in WT ovules at the MMC stage. (F-H) Signal 638

corresponding to pKNU:KNU-Venus was detected in two or three cells in mir390 (F), 639

zip-2 (G) and ago7-9 (H) MMC-stage ovules. (I) AGO9 immunolocalization in WT 640

pre-meiotic ovules. (J-L) AGO9 immunolocalization in mir390 (J), zip-2 (K) and 641

ago7-9 (L) pre-meiotic ovules. (M) Statistical tests of ovules showing multiple MMCs 642

in the mutants. (N) Statistical tests of ovules showing KNU-Venus in multiple cells. 643

(O) Statistical tests of ovules showing nuclear AGO9 immunolocalization signal in644

multiple cells. MMC-like cells are indicated by white arrows in (B-D). FM4-64 645

staining in (E-H) and PI staining in (I-L) are shown in magenta. The numbers in each 646

figure panel indicate the number of ovules with the phenotype shown in the image out 647

of the total number of ovules examined. Scale bars, 10 µm. Significant evaluations 648

between WT and mutants were performed by Student’s t-test (*p<0.05) in M-O. 649

650

Figure 2. Only one MMC undergoes meiosis in mir390 and ago7 ovules. (A–D) 651

DMC1 immunolocalization in pre-meiotic WT (A), mir390 (B), zip-2 (C) and ago7-9 652

(D) ovules. Propidium iodide staining in (A-D) is shown in magenta. (E-L) Callose653

deposition in WT (E, I), mir390 (F, J), zip-2 (G, K) and ago7-9 (H, L) ovules. (I-L) 654

Callose staining with aniline blue. (E-H) Morphology of the ovules in (I-L). 655

20

Abnormally enlarged cells in (B-D) and (J-L) are outlined with white dashed lines. 656

The numbers in each figure panel indicate the number of ovules with the phenotype 657

shown in the image out of the total number of ovules examined. Scale bars, 10 µm. 658

659

Figure 3. MIR390, AGO7 and SGS3 expression patterns in ovules. 660

(A) GUS signal in a pMIR390A:GUS ovule. Dashed white lines mark the boundary661

between the nucellus (nu), chalaza (ch) and funiculus (fu). (B) In situ localization of 662

miR390 in a longitudinal section through a WT ovule. The white lines represent the 663

positions of transverse sections in (D) and (E). (D,E) In situ localization of miR390 in 664

transverse sections of nucellus (D) and chalaza (E) of WT ovules. In (D), the position 665

of the section corresponds to the nucellus region as indicated by line “a” in (B). In (E), 666

the position of the section corresponds to the chalaza region as indicated by line “b” 667

in (B). (C,F) No signals were detected in longitudinal (C) and transverse (F) sections 668

of wild type ovules incubated without probe. (G,H) GFP signal in a 669

pAGO7:GFP-AGO7 ovule. GFP fluorescence is shown in (G); (H) shows the same 670

ovule as in (G) but with FM4-64 staining. (I) GUS signal in a pAGO7:GUS ovule. 671

(J-L) GFP signal in a pSGS3:SGS3-GFP ovule. GFP fluorescence is shown in (J); (K) 672

shows the same ovule as in (J) but with FM4-64 staining. (J) and (K) are merged in 673

(L). Scale bars, 10 µm. 674

675

Figure 4. AGO7 expression driven by pSPL rescued the multiple-MMC phenotype in 676

ago7. 677

(A,B) GFP signal in a pWRKY28:GFP-AGO7 ovule. GFP fluorescence is shown in 678

(A); (B) shows the same ovule as in (A) but also with FM4-64 staining. (C) A 679

pWRKY28:GFP-AGO7 ovule with two MMCs. MMC-like cells are indicated by 680

white arrows. (D-E) GFP signal in a pSPL:GFP-AGO7 ovule. GFP fluorescence is 681

shown in (D); (E) shows the same ovule as in (D) but also with FM4-64 staining. (F) 682

A pSPL:GFP-AGO7 ovule with one MMC. Scale bars, 10 µm. 683

684

Figure 5. ARF3 in situ hybridization in ovules. 685

(A-C) Whole-mount in situ hybridization of ARF3 in early MMC-stage WT (A) and 686

rdr6-11 (B) ovules. (C) WT ovule incubated with sense probe. (D-F) Whole-mount in 687

situ hybridization of ARF3 in later MMC-stage WT (D) and rdr6-11 (H) ovules. (F) 688

21

WT ovule incubated without probe. Dashed red lines (A,B,D and E) mark the 689

boundary between the nucellus and chalaza. Bars, 10 µm. 690

691

Figure 6. ARF3m expression driven by pWRKY28 resulted in a multiple-MMC 692

phenotype. 693

(A-C) ARF3m-mCitrine signals in pSPL:ARF3m-mCitrine ovules. (D) A 694

pSPL:ARF3m-mCitrine ovule with one MMC. (E-G) ARF3m-mCitrine signals in 695

pWRKY28:ARF3m-mCitrine ovules. (H) A pWRKY28:ARF3m-mCitrine ovule with 696

two MMC-like cells. (I-K) GUS activity corresponding to pKNU:nlsGUS expression 697

in WT (I) and pWRKY28:ARF3m-mCitrine ovules (J, K). (L) AGO9 698

immunolocalization in pWRKY28:ARF3m-mCitrine pre-meiotic ovules. mCitrine 699

fluorescence is shown in (A) and (E); (B) and (F) show the same ovules as in the 700

panels to their left but with FM4-64 staining. (A) and (B) are merged in (C), and (E) 701

and (F) are merged in (G). The MMC is marked with white dashed lines in (C) and 702

(G). The MMC-like cells in (H-L) are indicated by white arrows. Bars, 10 µm. 703

704

Figure 7. A model of tasiR-ARF biogenesis and mobility. 705

(A) TasiR-ARFs are processed in the apical epidermal cells (labeled in yellow) and706

move to hypodermal cells in the nucellar region to restrain ARF3 expression and 707

suppress MMC identity. The tasiR-ARFs also move basipetally from the nucellus into 708

the chalaza. The distribution of tasiR-ARF3 and ARF3 RNA are labeled in green and 709

pink, respectively. (B) Without the control by tasiR-ARFs, ARF3 expression expands 710

both acropetally and radially. The expression of ARF3 in the hypodermal cells 711

(labeled with asterisks) leads to MMC characteristics. 712

713

References 714

715

Allen, E., Xie, Z., Gustafson, A.M., and Carrington, J.C. (2005). microRNA-directed phasing during 716

trans-acting siRNA biogenesis in plants. Cell 121:207-221. 717

Baumberger, N., and Baulcombe, D.C. (2005). Arabidopsis ARGONAUTE1 is an RNA Slicer that 718

selectively recruits rnicroRNAs and short interfering RNAs. Proceedings of the National 719

Academy of Sciences of the United States of America 102:11928-11933. 720

Bicknell, R.A., and Koltunow, A.M. (2004). Understanding apomixis: recent advances and remaining 721

conundrums. The Plant cell 16 Suppl:S228-245. 722

Cao, L., Wang, S., Venglat, P., Zhao, L., Cheng, Y., Ye, S., Qin, Y., Datla, R., Zhou, Y., and Wang, H. 723

(2018). Arabidopsis ICK/KRP cyclin-dependent kinase inhibitors function to ensure the 724

22

formation of one megaspore mother cell and one functional megaspore per ovule. PLoS 725

genetics 14:e1007230. 726

Chen, C., Xia, R., Chen, H., and He, Y.J.B. (2018). TBtools, a Toolkit for Biologists integrating various 727

biological data handling tools with a user-friendly interface.289660. 728

Chen, X.M. (2009). Small RNAs and Their Roles in Plant Development. Annu Rev Cell Dev Bi 729

25:21-44. 730

Chitwood, D.H., Nogueira, F.T., Howell, M.D., Montgomery, T.A., Carrington, J.C., and Timmermans, 731

M.C. (2009). Pattern formation via small RNA mobility. Genes & development 23:549-554. 732

Cuperus, J.T., Montgomery, T.A., Fahlgren, N., Burke, R.T., Townsend, T., Sullivan, C.M., and 733

Carrington, J.C. (2010). Identification of MIR390a precursor processing-defective mutants in 734

Arabidopsis by direct genome sequencing. Proceedings of the National Academy of Sciences 735

of the United States of America 107:466-471. 736

Curtis, M.D., and Grossniklaus, U. (2003). A gateway cloning vector set for high-throughput functional 737

analysis of genes in planta. Plant physiology 133:462-469. 738

Dotto, M.C., Petsch, K.A., Aukerman, M.J., Beatty, M., Hammell, M., and Timmermans, M.C. (2014). 739

Genome-wide analysis of leafbladeless1-regulated and phased small RNAs underscores the 740

importance of the TAS3 ta-siRNA pathway to maize development. PLoS genetics 741

10:e1004826. 742

Endo, Y., Iwakawa, H.O., and Tomari, Y. (2013). Arabidopsis ARGONAUTE7 selects miR390 through 743

multiple checkpoints during RISC assembly. EMBO Rep 14:652-658. 744

Escobar-Guzman, R., Rodriguez-Leal, D., Vielle-Calzada, J.P., and Ronceret, A. (2015). Whole-mount 745

immunolocalization to study female meiosis in Arabidopsis. Nature protocols 10:1535-1542. 746

Fahlgren, N., Montgomery, T.A., Howell, M.D., Allen, E., Dvorak, S.K., Alexander, A.L., and 747

Carrington, J.C. (2006). Regulation of AUXIN RESPONSE FACTOR3 by TAS3 ta-siRNA 748

affects developmental timing and patterning in Arabidopsis. Current biology : CB 16:939-944. 749

Ferreira, J.A., and Nyangoma, S.O.J.J.o.M.A. (2008). A multivariate version of the Benjamini–750

Hochberg method. 99:2108-2124. 751

Garcia, D., Collier, S.A., Byrne, M.E., and Martienssen, R.A. (2006). Specification of leaf polarity in 752

Arabidopsis via the trans-acting siRNA pathway. Current Biology 16:933-938. 753

Gasciolli, V., Mallory, A.C., Bartel, D.P., and Vaucheret, H. (2005). Partially redundant functions of 754

Arabidopsis DICER-like enzymes and a role for DCL4 in producing trans-acting siRNAs. 755

Current Biology 15:1494-1500. 756

Gross-Hardt, R., Lenhard, M., and Laux, T. (2002). WUSCHEL signaling functions in interregional 757

communication during Arabidopsis ovule development. Genes & development 16:1129-1138. 758

Grossniklaus, U. (2011). Plant germline development: a tale of cross-talk, signaling, and cellular 759

interactions. Sex Plant Reprod 24:91-95. 760

Hejatko, J., Blilou, I., Brewer, P.B., Friml, J., Scheres, B., and Benkova, E. (2006). In situ hybridization 761

technique for mRNA detection in whole mount Arabidopsis samples. Nature protocols 762

1:1939-1946. 763

Hunter, C., Willmann, M.R., Wu, G., Yoshikawa, M., de la Luz Gutierrez-Nava, M., and Poethig, S.R. 764

(2006). Trans-acting siRNA-mediated repression of ETTIN and ARF4 regulates heteroblasty 765

in Arabidopsis. Development 133:2973-2981. 766

Imran Siddiqi, G.G., Ueli Grossniklaus, Veeraputhiran Subbiah. (2000). The dyad gene is required for 767

progression through female meiosis in Arabidopsis. Development:11. 768

23

Jauvion, V., Elmayan, T., and Vaucheret, H. (2010). The Conserved RNA Trafficking Proteins HPR1 769

and TEX1 Are Involved in the Production of Endogenous and Exogenous Small Interfering 770

RNA in Arabidopsis. The Plant cell 22:2697-2709. 771

Kidner, C., and Timmermans, M. (2006). In situ hybridization as a tool to study the role of microRNAs 772

in plant development. Methods in molecular biology 342:159-179. 773

Klimyuk, V.I., and Jones, J.D. (1997). AtDMC1, the Arabidopsis homologue of the yeast DMC1 gene: 774

characterization, transposon-induced allelic variation and meiosis-associated expression. The 775

Plant journal : for cell and molecular biology 11:1-14. 776

Li, L., Wu, W., Zhao, Y., and Zheng, B. (2017). A reciprocal inhibition between ARID1 and MET1 in 777

male and female gametes in Arabidopsis. Journal of integrative plant biology 59:657-668. 778

Li, X., Lei, M., Yan, Z., Wang, Q., Chen, A., Sun, J., Luo, D., and Wang, Y. (2014). The 779

REL3-mediated TAS3 ta-siRNA pathway integrates auxin and ethylene signaling to regulate 780

nodulation in Lotus japonicus. New Phytol 201:531-544. 781

Liu, X., Dinh, T.T., Li, D., Shi, B., Li, Y., Cao, X., Guo, L., Pan, Y., Jiao, Y., and Chen, X. (2014). 782

AUXIN RESPONSE FACTOR 3 integrates the functions of AGAMOUS and APETALA2 in 783

floral meristem determinacy. The Plant journal : for cell and molecular biology 80:629-641. 784

Luo, Q.J., Mittal, A., Jia, F., and Rock, C.D. (2012). An autoregulatory feedback loop involving PAP1 785

and TAS4 in response to sugars in Arabidopsis. Plant Mol Biol 80:117-129. 786

Marin, E., Jouannet, V., Herz, A., Lokerse, A.S., Weijers, D., Vaucheret, H., Nussaume, L., Crespi, 787

M.D., and Maizel, A. (2010). miR390, Arabidopsis TAS3 tasiRNAs, and Their AUXIN788

RESPONSE FACTOR Targets Define an Autoregulatory Network Quantitatively Regulating 789

Lateral Root Growth. The Plant cell 22:1104-1117. 790

Mary C. Webb, B.E.S.G. (1990). Embryo sac development in Arabidopsis thaliana. Sex Plant Reprod 791

3:13. 792

Montgomery, T.A., Howell, M.D., Cuperus, J.T., Li, D., Hansen, J.E., Alexander, A.L., Chapman, E.J., 793

Fahlgren, N., Allen, E., and Carrington, J.C. (2008a). Specificity of ARGONAUTE7-miR390 794

interaction and dual functionality in TAS3 trans-acting siRNA formation. Cell 133:128-141. 795

Montgomery, T.A., Yoo, S.J., Fahlgren, N., Gilbert, S.D., Howell, M.D., Sullivan, C.M., Alexander, A., 796

Nguyen, G., Allen, E., Ahn, J.H., et al. (2008b). AGO1-miR173 complex initiates phased 797

siRNA formation in plants. Proceedings of the National Academy of Sciences of the United 798

States of America 105:20055-20062. 799

Nakagawa, T., Kurose, T., Hino, T., Tanaka, K., Kawamukai, M., Niwa, Y., Toyooka, K., Matsuoka, K., 800

Jinbo, T., and Kimura, T. (2007). Development of series of gateway binary vectors, pGWBs, 801

for realizing efficient construction of fusion genes for plant transformation. J Biosci Bioeng 802

104:34-41. 803

Nogueira, F.T., Madi, S., Chitwood, D.H., Juarez, M.T., and Timmermans, M.C. (2007). Two small 804

regulatory RNAs establish opposing fates of a developmental axis. Genes & development 805

21:750-755. 806

Oh, S.A., Pal, M.D., Park, S.K., Johnson, J.A., and Twell, D. (2010). The tobacco 807

MAP215/Dis1-family protein TMBP200 is required for the functional organization of 808

microtubule arrays during male germline establishment. J Exp Bot 61:969-981. 809

Olmedo-Monfil, V., Duran-Figueroa, N., Arteaga-Vazquez, M., Demesa-Arevalo, E., Autran, D., 810

Grimanelli, D., Slotkin, R.K., Martienssen, R.A., and Vielle-Calzada, J.P. (2010). Control of 811

female gamete formation by a small RNA pathway in Arabidopsis. Nature 464:628-632. 812

24

Ozerova, L.V., Krasnikova, M.S., Troitsky, A.V., Solovyev, A.G., and Morozov, S.Y. (2013). TAS3 813

Genes for small ta-siARF RNAs in plants belonging to subtribe Senecioninae: occurrence of 814

prematurely terminated RNA precursors. Mol Gen Mikrobiol Virusol:33-36. 815

Payne, T., Johnson, S.D., and Koltunow, A.M. (2004). KNUCKLES (KNU) encodes a C2H2 816

zinc-finger protein that regulates development of basal pattern elements of the Arabidopsis 817

gynoecium. Development 131:3737-3749. 818

Peragine, A., Yoshikawa, M., Wu, G., Albrecht, H.L., and Poethig, R.S. (2004). SGS3 and 819

SGS2/SDE1/RDR6 are required for juvenile development and the production of trans-acting 820

siRNAs in Arabidopsis. Genes & development 18:2368-2379. 821

Rajagopalan, R., Vaucheret, H., Trejo, J., and Bartel, D.P. (2006). A diverse and evolutionarily fluid set 822

of microRNAs in Arabidopsis thaliana. Genes & development 20:3407-3425. 823

Robinson-Beers, K., Pruitt, R.E., and Gasser, C.S. (1992). Ovule Development in Wild-Type 824

Arabidopsis and Two Female-Sterile Mutants. The Plant cell 4:1237-1249. 825

Rodriguez-Leal, D., Leon-Martinez, G., Abad-Vivero, U., and Vielle-Calzada, J.P. (2015). Natural 826

variation in epigenetic pathways affects the specification of female gamete precursors in 827

Arabidopsis. The Plant cell 27:1034-1045. 828

Schiefthaler, U., Balasubramanian, S., Sieber, P., Chevalier, D., Wisman, E., and Schneitz, K. (1999). 829

Molecular analysis of NOZZLE, a gene involved in pattern formation and early sporogenesis 830

during sex organ development in Arabidopsis thaliana. Proceedings of the National Academy 831

of Sciences of the United States of America 96:11664-11669. 832

Schmidt, A., Wuest, S.E., Vijverberg, K., Baroux, C., Kleen, D., and Grossniklaus, U. (2011). 833

Transcriptome analysis of the Arabidopsis megaspore mother cell uncovers the importance of 834

RNA helicases for plant germline development. PLoS biology 9:e1001155. 835

Shirley, N.J., Aubert, M.K., Wilkinson, L.G., Bird, D.C., Lora, J., Yang, X.J., and Tucker, M.R. (2019). 836

Translating auxin responses into ovules, seeds and yield: Insight from Arabidopsis and the 837

cereals. Journal of integrative plant biology 61:310-336. 838

Sieber, P., Gheyselinck, J., Gross-Hardt, R., Laux, T., Grossniklaus, U., and Schneitz, K. (2004). 839

Pattern formation during early ovule development in Arabidopsis thaliana. Dev Biol 840

273:321-334. 841

Simonini, S., Bencivenga, S., Trick, M., and Ostergaard, L. (2017). Auxin-Induced Modulation of 842

ETTIN Activity Orchestrates Gene Expression in Arabidopsis. The Plant cell 29:1864-1882. 843

Singh, M., Goel, S., Meeley, R.B., Dantec, C., Parrinello, H., Michaud, C., Leblanc, O., and Grimanelli, 844

D. (2011). Production of viable gametes without meiosis in maize deficient for an 845

ARGONAUTE protein. The Plant cell 23:443-458. 846

Singh, S.K., Kumar, V., Srinivasan, R., Ahuja, P.S., Bhat, S.R., and Sreenivasulu, Y. (2017). The TRAF 847

Mediated Gametogenesis Progression (TRAMGaP) Gene Is Required for Megaspore Mother 848

Cell Specification and Gametophyte Development. Plant physiology 175:1220-1237. 849

Su, Z., Zhao, L., Zhao, Y., Li, S., Won, S., Cai, H., Wang, L., Li, Z., Chen, P., Qin, Y., et al. (2017). The 850

THO Complex Non-Cell-Autonomously Represses Female Germline Specification through 851

the TAS3-ARF3 Module. Current biology : CB 27:1597-1609 e1592. 852

Vazquez, F., Vaucheret, H., Rajagopalan, R., Lepers, C., Gasciolli, V., Mallory, A.C., Hilbert, J.L., 853

Bartel, D.P., and Crete, P. (2004). Endogenous trans-acting siRNAs regulate the accumulation 854

of Arabidopsis mRNAs. Molecular cell 16:69-79. 855

Walbot, V., and Evans, M.M. (2003). Unique features of the plant life cycle and their consequences. 856

25

Nat Rev Genet 4:369-379. 857

Wang, J., Gao, X., Li, L., Shi, X., Zhang, J., and Shi, Z. (2010). Overexpression of Osta-siR2141 858

caused abnormal polarity establishment and retarded growth in rice. J Exp Bot 61:1885-1895. 859

Wei, B., Zhang, J., Pang, C., Yu, H., Guo, D., Jiang, H., Ding, M., Chen, Z., Tao, Q., Gu, H., et al. 860

(2015). The molecular mechanism of sporocyteless/nozzle in controlling Arabidopsis ovule 861

development. Cell research 25:121-134. 862

Williams, L., Carles, C.C., Osmont, K.S., and Fletcher, J.C. (2005). A database analysis method 863

identifies an endogenous trans-acting short-interfering RNA that targets the Arabidopsis ARF2, 864

ARF3, and ARF4 genes. Proceedings of the National Academy of Sciences of the United 865

States of America 102:9703-9708. 866

Xia, R., Xu, J., and Meyers, B.C. (2017). The Emergence, Evolution, and Diversification of the 867

miR390-TAS3-ARF Pathway in Land Plants. The Plant cell 29:1232-1247. 868

Xie, Z.X., Allen, E., Wilken, A., and Carrington, J.C. (2005). DICER-LIKE 4 functions in trans-acting 869

small interfering RNA biogenesis and vegetative phase change in Arabidopsis thaliana. 870

Proceedings of the National Academy of Sciences of the United States of America 871

102:12984-12989. 872

Yang, W.C., Shi, D.Q., and Chen, Y.H. (2010). Female gametophyte development in flowering plants. 873

Annual review of plant biology 61:89-108. 874

Yang, W.C., Ye, D., Xu, J., and Sundaresan, V. (1999). The SPOROCYTELESS gene of Arabidopsis is 875

required for initiation of sporogenesis and encodes a novel nuclear protein. Genes & 876

development 13:2108-2117. 877

Yao, X., Yang, H., Zhu, Y., Xue, J., Wang, T., Song, T., Yang, Z., and Wang, S. (2018). The Canonical 878

E2Fs Are Required for Germline Development in Arabidopsis. Frontiers in plant science 879

9:638. 880

Yelina, N.E., Smith, L.M., Jones, A.M., Patel, K., Kelly, K.A., and Baulcombe, D.C. (2010). Putative 881

Arabidopsis THO/TREX mRNA export complex is involved in transgene and endogenous 882

siRNA biosynthesis. Proceedings of the National Academy of Sciences of the United States of 883

America 107:13948-13953. 884

Yoshikawa, M., Iki, T., Tsutsui, Y., Miyashita, K., Poethig, R.S., Habu, Y., and Ishikawa, M. (2013). 3' 885

fragment of miR173-programmed RISC-cleaved RNA is protected from degradation in a 886

complex with RISC and SGS3. Proceedings of the National Academy of Sciences of the 887

United States of America 110:4117-4122. 888

Yoshikawa, M., Peragine, A., Park, M.Y., and Poethig, R.S. (2005). A pathway for the biogenesis of 889

trans-acting siRNAs in Arabidopsis. Genes & development 19:2164-2175. 890

Zhao, L., Cai, H., Su, Z., Wang, L., Huang, X., Zhang, M., Chen, P., Dai, X., Zhao, H., Palanivelu, R., 891

et al. (2018). KLU suppresses megasporocyte cell fate through SWR1-mediated activation of 892

WRKY28 expression in Arabidopsis. Proceedings of the National Academy of Sciences of the 893

United States of America 115:E526-E535. 894

Zheng, Y., Zhang, K., Guo, L., Liu, X., and Zhang, Z. (2018). AUXIN RESPONSE FACTOR3 plays 895

distinct role during early flower development. Plant Signal Behav 13:e1467690. 896

Figure 1. MMC-like cells form in mir390 and ago7 ovules. (A) WT ovule with one MMC. (B-D) Two MMC-like cells in a mir390 ovule (B), azip-2 ovule (C) and an ago7-9 ovule (D). (E) Signal corresponding topKNU:KNU-Venus was detected in one cell in WT ovules at the MMC stage. (F-H)Signal corresponding to pKNU:KNU-Venus was detected in two or three cells inmir390 (F), zip-2 (G) and ago7-9 (H) MMC-stage ovules. (I) AGO9immunolocalization in WT pre-meiotic ovules. (J-L) AGO9 immunolocalization inmir390 (J), zip-2 (K) and ago7-9 (L) pre-meiotic ovules. (M) Statistical tests of ovulesshowing multiple MMCs in the mutants. (N) Statistical tests of ovules showingKNU-Venus in multiple cells. (O) Statistical tests of ovules showing nuclear AGO9immunolocalization signal in multiple cells. MMC-like cells are indicated by whitearrows in (B-D). FM4-64 staining in (E-H) and PI staining in (I-L) are shown inmagenta. The numbers in each figure panel indicate the number of ovules with thephenotype shown in the image out of the total number of ovules examined. Scale bars,10 µm. Significant evaluations between WT and mutants were performed by student’st-test (*p<0.05) in M-O.

Figure 2. Only one MMC undergoes meiosis in mir390 and ago7 ovules. (A–D) DMC1 immunolocalization in pre-meiotic WT (A), mir390 (B), zip-2 (C) and ago7-9 (D) ovules. Propidium iodide staining in (A-D) is shown in magenta. (E-L) Callose deposition in WT (E, I), mir390 (F, J), zip-2 (G, K) and ago7-9 (H, L) ovules. (I-L) Callose staining with aniline blue. (E-H) Morphology of the ovules in (I-L). Abnormally enlarged cells in (B-D) and (J-L) are outlined with white dashed lines. The numbers in each figure panel indicate the number of ovules with the phenotype shown in the image out of the total number of ovules examined. Scale bars, 10 µm.

Figure 3. MIR390, AGO7 and SGS3 expression patterns in ovules. (A) GUS signal in a pMIR390A:GUS ovule. Dashed white lines mark the boundarybetween the nucellus (nu), chalaza (ch) and funiculus (fu). (B) In situ localization ofmiR390 in a longitudinal section through a WT ovule. The white lines represent thepositions of transverse sections in (D) and (E). (D,E) In situ localization of miR390 intransverse sections of nucellus (D) and chalaza (E) of WT ovules. In (D), the positionof the section corresponds to the nucellus region as indicated by line “a” in (B). In (E),the position of the section corresponds to the chalaza region as indicated by line “b”in (B). (C,F) No signals were detected in longitudinal (C) and transverse (F) sectionsof wild type ovules incubated without probe. (G,H) GFP signal in apAGO7:GFP-AGO7 ovule. GFP fluorescence is shown in (G); (H) shows the sameovule as in (G) but with FM4-64 staining. (I) GUS signal in a pAGO7:GUS ovule.(J-L) GFP signal in a pSGS3:SGS3-GFP ovule. GFP fluorescence is shown in (J); (K)shows the same ovule as in (J) but with FM4-64 staining. (J) and (K) are merged in(L). Scale bars, 10 µm.

Figure 4. AGO7 expression driven by pSPL rescued the multiple-MMC phenotype in ago7. (A,B) GFP signal in a pWRKY28:GFP-AGO7 ovule. GFP fluorescence is shown in (A); (B) shows the same ovule as in (A) but also with FM4-64 staining. (C) A pWRKY28:GFP-AGO7 ovule with two MMCs. MMC-like cells are indicated by white arrows. (D-E) GFP signal in a pSPL:GFP-AGO7 ovule. GFP fluorescence is shown in (D); (E) shows the same ovule as in (D) but also with FM4-64 staining. (F) A pSPL:GFP-AGO7 ovule with one MMC. Scale bars, 10 µm.

Figure 5. ARF3 in situ hybridization in ovules. (A-C) Whole-mount in situ hybridization of ARF3 in early MMC-stage WT (A) and rdr6-11 (B) ovules. (C) WT ovule incubated with sense probe. (D-F) Whole-mount in situ hybridization of ARF3 in later MMC-stage WT (D) and rdr6-11 (H) ovules. (F) WT ovule incubated without probe. Dashed red lines (A,B,D and E) mark the boundary between the nucellus and chalaza. Bars, 10 µm.

Figure 6. ARF3m expression driven by pWRKY28 resulted in a multiple-MMC phenotype. (A-C) ARF3m-mCitrine signals in pSPL:ARF3m-mCitrine ovules. (D) A pSPL:ARF3m-mCitrine ovule with one MMC. (E-G) ARF3m-mCitrine signals in pWRKY28:ARF3m-mCitrine ovules. (H) A pWRKY28:ARF3m-mCitrine ovule with two MMC-like cells. (I-K) GUS activity corresponding to pKNU:nlsGUS expression in WT (I) and pWRKY28:ARF3m-mCitrine ovules (J, K). (L) AGO9 immunolocalization in pWRKY28:ARF3m-mCitrine pre-meiotic ovules. mCitrine fluorescence is shown in (A) and (E); (B) and (F) show the same ovules as in the panels to their left but with FM4-64 staining. (A) and (B) are merged in (C), and (E) and (F) are merged in (G). The MMC is marked with white dashed lines in (C) and (G). The MMC-like cells in (H-L) are indicated by white arrows. Bars, 10 µm.

Figure 7. A model of tasiR-ARF biogenesis and mobility. (A) TasiR-ARFs are processed in the apical epidermal cells (labeled in yellow) andmove to hypodermal cells in the nucellar region to restrain ARF3 expression andsuppress MMC identity. The tasiR-ARFs also move basipetally from the nucellus intothe chalaza. The distribution of tasiR-ARF3 and ARF3 RNA are labeled in green andpink, respectively. (B) Without the control by tasiR-ARFs, ARF3 expression expandsboth acropetally and radially. The expression of ARF3 in the hypodermal cells(labeled with asterisks) leads to MMC characteristics.

Parsed CitationsAllen, E., Xie, Z., Gustafson, A.M., and Carrington, J.C. (2005). microRNA-directed phasing during trans-acting siRNA biogenesis inplants. Cell 121:207-221.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Baumberger, N., and Baulcombe, D.C. (2005). Arabidopsis ARGONAUTE1 is an RNA Slicer that selectively recruits rnicroRNAs andshort interfering RNAs. Proceedings of the National Academy of Sciences of the United States of America 102:11928-11933.


Bicknell, R.A., and Koltunow, A.M. (2004). Understanding apomixis: recent advances and remaining conundrums. The Plant cell 16Suppl:S228-245.


Cao, L., Wang, S., Venglat, P., Zhao, L., Cheng, Y., Ye, S., Qin, Y., Datla, R., Zhou, Y., and Wang, H. (2018). Arabidopsis ICK/KRP cyclin-dependent kinase inhibitors function to ensure the formation of one megaspore mother cell and one functional megaspore per ovule.PLoS genetics 14:e1007230.


Chen, C., Xia, R., Chen, H., and He, Y.J.B. (2018). TBtools, a Toolkit for Biologists integrating various biological data handling toolswith a user-friendly interface.289660.


Chen, X.M. (2009). Small RNAs and Their Roles in Plant Development. Annu Rev Cell Dev Bi 25:21-44.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Chitwood, D.H., Nogueira, F.T., Howell, M.D., Montgomery, T.A., Carrington, J.C., and Timmermans, M.C. (2009). Pattern formation viasmall RNA mobility. Genes & development 23:549-554.


Cuperus, J.T., Montgomery, T.A., Fahlgren, N., Burke, R.T., Townsend, T., Sullivan, C.M., and Carrington, J.C. (2010). Identification ofMIR390a precursor processing-defective mutants in Arabidopsis by direct genome sequencing. Proceedings of the National Academyof Sciences of the United States of America 107:466-471.


Curtis, M.D., and Grossniklaus, U. (2003). A gateway cloning vector set for high-throughput functional analysis of genes in planta. Plantphysiology 133:462-469.


Dotto, M.C., Petsch, K.A., Aukerman, M.J., Beatty, M., Hammell, M., and Timmermans, M.C. (2014). Genome-wide analysis ofleafbladeless1-regulated and phased small RNAs underscores the importance of the TAS3 ta-siRNA pathway to maize development.PLoS genetics 10:e1004826.


Endo, Y., Iwakawa, H.O., and Tomari, Y. (2013). Arabidopsis ARGONAUTE7 selects miR390 through multiple checkpoints during RISCassembly. EMBO Rep 14:652-658.


Escobar-Guzman, R., Rodriguez-Leal, D., Vielle-Calzada, J.P., and Ronceret, A. (2015). Whole-mount immunolocalization to study femalemeiosis in Arabidopsis. Nature protocols 10:1535-1542.


Fahlgren, N., Montgomery, T.A., Howell, M.D., Allen, E., Dvorak, S.K., Alexander, A.L., and Carrington, J.C. (2006). Regulation of AUXINRESPONSE FACTOR3 by TAS3 ta-siRNA affects developmental timing and patterning in Arabidopsis. Current biology : CB 16:939-944.


Ferreira, J.A., and Nyangoma, S.O.J.J.o.M.A. (2008). A multivariate version of the Benjamini–Hochberg method. 99:2108-2124.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Garcia, D., Collier, S.A., Byrne, M.E., and Martienssen, R.A. (2006). Specification of leaf polarity in Arabidopsis via the trans-acting

https://www.ncbi.nlm.nih.gov/pubmed?term=Allen%2C E%2E%2C Xie%2C Z%2E%2C Gustafson%2C A%2EM%2E%2C and Carrington%2C J%2EC%2E %282005%29%2E microRNA%2Ddirected phasing during trans%2Dacting siRNA biogenesis in plants%2E Cell&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Allen,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=microRNA-directed phasing during trans-acting siRNA biogenesis in plants&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=microRNA-directed phasing during trans-acting siRNA biogenesis in plants&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Allen,&as_ylo=2005&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Baumberger%2C N%2E%2C and Baulcombe%2C D%2EC%2E %282005%29%2E Arabidopsis ARGONAUTE1 is an RNA Slicer that selectively recruits rnicroRNAs and short interfering RNAs%2E Proceedings of the National Academy of Sciences of the United States of America&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Baumberger,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Arabidopsis ARGONAUTE1 is an RNA Slicer that selectively recruits rnicroRNAs and short interfering RNAs&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Arabidopsis ARGONAUTE1 is an RNA Slicer that selectively recruits rnicroRNAs and short interfering RNAs&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Baumberger,&as_ylo=2005&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Bicknell%2C R%2EA%2E%2C and Koltunow%2C A%2EM%2E %282004%29%2E Understanding apomixis%3A recent advances and remaining conundrums%2E The Plant cell 16 Suppl%3AS&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Bicknell,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Bicknell,&as_ylo=2004&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Cao%2C L%2E%2C Wang%2C S%2E%2C Venglat%2C P%2E%2C Zhao%2C L%2E%2C Cheng%2C Y%2E%2C Ye%2C S%2E%2C Qin%2C Y%2E%2C Datla%2C R%2E%2C Zhou%2C Y%2E%2C and Wang%2C H%2E %282018%29%2E Arabidopsis ICK%2FKRP cyclin%2Ddependent kinase inhibitors function to ensure the formation of one megaspore mother cell and one functional megaspore per ovule%2E PLoS genetics 14%3Ae&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Cao,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Arabidopsis ICK/KRP cyclin-dependent kinase inhibitors function to ensure the formation of one megaspore mother cell and one functional megaspore per ovule&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Arabidopsis ICK/KRP cyclin-dependent kinase inhibitors function to ensure the formation of one megaspore mother cell and one functional megaspore per ovule&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Cao,&as_ylo=2018&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Chen%2C C%2E%2C Xia%2C R%2E%2C Chen%2C H%2E%2C and He%2C Y%2EJ%2EB%2E %282018%29%2E TBtools%2C a Toolkit for Biologists integrating various biological data handling tools with a user%2Dfriendly interface&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Chen,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Xia, R&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Xia, R&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Chen,&as_ylo=9660.&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Chen%2C X%2EM%2E %282009%29%2E Small RNAs and Their Roles in Plant Development%2E Annu Rev Cell Dev Bi&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Chen,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Small RNAs and Their Roles in Plant Development&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Small RNAs and Their Roles in Plant Development&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Chen,&as_ylo=2009&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Chitwood%2C D%2EH%2E%2C Nogueira%2C F%2ET%2E%2C Howell%2C M%2ED%2E%2C Montgomery%2C T%2EA%2E%2C Carrington%2C J%2EC%2E%2C and Timmermans%2C M%2EC%2E %282009%29%2E Pattern formation via small RNA mobility%2E Genes %26 development&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Chitwood,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Pattern formation via small RNA mobility&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Pattern formation via small RNA mobility&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Chitwood,&as_ylo=2009&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Cuperus%2C J%2ET%2E%2C Montgomery%2C T%2EA%2E%2C Fahlgren%2C N%2E%2C Burke%2C R%2ET%2E%2C Townsend%2C T%2E%2C Sullivan%2C C%2EM%2E%2C and Carrington%2C J%2EC%2E %282010%29%2E Identification of MIR390a precursor processing%2Ddefective mutants in Arabidopsis by direct genome sequencing%2E Proceedings of the National Academy of Sciences of the United States of America&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Cuperus,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Identification of MIR390a precursor processing-defective mutants in Arabidopsis by direct genome sequencing&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Identification of MIR390a precursor processing-defective mutants in Arabidopsis by direct genome sequencing&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Cuperus,&as_ylo=2010&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Curtis%2C M%2ED%2E%2C and Grossniklaus%2C U%2E %282003%29%2E A gateway cloning vector set for high%2Dthroughput functional analysis of genes in planta%2E Plant physiology&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Curtis,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=A gateway cloning vector set for high-throughput functional analysis of genes in planta&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=A gateway cloning vector set for high-throughput functional analysis of genes in planta&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Curtis,&as_ylo=2003&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Dotto%2C M%2EC%2E%2C Petsch%2C K%2EA%2E%2C Aukerman%2C M%2EJ%2E%2C Beatty%2C M%2E%2C Hammell%2C M%2E%2C and Timmermans%2C M%2EC%2E %282014%29%2E Genome%2Dwide analysis of leafbladeless1%2Dregulated and phased small RNAs underscores the importance of the TAS3 ta%2DsiRNA pathway to maize development%2E PLoS genetics 10%3Ae&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Dotto,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Genome-wide analysis of leafbladeless1-regulated and phased small RNAs underscores the importance of the TAS3 ta-siRNA pathway to maize development&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Genome-wide analysis of leafbladeless1-regulated and phased small RNAs underscores the importance of the TAS3 ta-siRNA pathway to maize development&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Dotto,&as_ylo=2014&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Endo%2C Y%2E%2C Iwakawa%2C H%2EO%2E%2C and Tomari%2C Y%2E %282013%29%2E Arabidopsis ARGONAUTE7 selects miR390 through multiple checkpoints during RISC assembly%2E EMBO Rep&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Endo,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Arabidopsis ARGONAUTE7 selects miR390 through multiple checkpoints during RISC assembly&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Arabidopsis ARGONAUTE7 selects miR390 through multiple checkpoints during RISC assembly&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Endo,&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Escobar%2DGuzman%2C R%2E%2C Rodriguez%2DLeal%2C D%2E%2C Vielle%2DCalzada%2C J%2EP%2E%2C and Ronceret%2C A%2E %282015%29%2E Whole%2Dmount immunolocalization to study female meiosis in Arabidopsis%2E Nature protocols&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Escobar-Guzman,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Whole-mount immunolocalization to study female meiosis in Arabidopsis&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Whole-mount immunolocalization to study female meiosis in Arabidopsis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Escobar-Guzman,&as_ylo=2015&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Fahlgren%2C N%2E%2C Montgomery%2C T%2EA%2E%2C Howell%2C M%2ED%2E%2C Allen%2C E%2E%2C Dvorak%2C S%2EK%2E%2C Alexander%2C A%2EL%2E%2C and Carrington%2C J%2EC%2E %282006%29%2E Regulation of AUXIN RESPONSE FACTOR3 by TAS3 ta%2DsiRNA affects developmental timing and patterning in Arabidopsis%2E Current biology %3A CB&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Fahlgren,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Regulation of AUXIN RESPONSE FACTOR3 by TAS3 ta-siRNA affects developmental timing and patterning in Arabidopsis&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Regulation of AUXIN RESPONSE FACTOR3 by TAS3 ta-siRNA affects developmental timing and patterning in Arabidopsis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Fahlgren,&as_ylo=2006&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Ferreira%2C J%2EA%2E%2C and Nyangoma%2C S%2EO%2EJ%2EJ%2Eo%2EM%2EA%2E %282008%29%2E A multivariate version of the Benjamini%26%23x2013%3BHochberg method&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Ferreira,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=A multivariate version of the Benjamini?Hochberg method&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=A multivariate version of the Benjamini?Hochberg method&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Ferreira,&as_ylo=2008&as_allsubj=all&hl=en&c2coff=1

siRNA pathway. Current Biology 16:933-938.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Gasciolli, V., Mallory, A.C., Bartel, D.P., and Vaucheret, H. (2005). Partially redundant functions of Arabidopsis DICER-like enzymes anda role for DCL4 in producing trans-acting siRNAs. Current Biology 15:1494-1500.


Gross-Hardt, R., Lenhard, M., and Laux, T. (2002). WUSCHEL signaling functions in interregional communication during Arabidopsisovule development. Genes & development 16:1129-1138.


Grossniklaus, U. (2011). Plant germline development: a tale of cross-talk, signaling, and cellular interactions. Sex Plant Reprod 24:91-95.


Hejatko, J., Blilou, I., Brewer, P.B., Friml, J., Scheres, B., and Benkova, E. (2006). In situ hybridization technique for mRNA detection inwhole mount Arabidopsis samples. Nature protocols 1:1939-1946.


Hunter, C., Willmann, M.R., Wu, G., Yoshikawa, M., de la Luz Gutierrez-Nava, M., and Poethig, S.R. (2006). Trans-acting siRNA-mediatedrepression of ETTIN and ARF4 regulates heteroblasty in Arabidopsis. Development 133:2973-2981.


Imran Siddiqi, G.G., Ueli Grossniklaus, Veeraputhiran Subbiah. (2000). The dyad gene is required for progression through femalemeiosis in Arabidopsis. Development:11.


Jauvion, V., Elmayan, T., and Vaucheret, H. (2010). The Conserved RNA Trafficking Proteins HPR1 and TEX1 Are Involved in theProduction of Endogenous and Exogenous Small Interfering RNA in Arabidopsis. The Plant cell 22:2697-2709.


Kidner, C., and Timmermans, M. (2006). In situ hybridization as a tool to study the role of microRNAs in plant development. Methods inmolecular biology 342:159-179.


Klimyuk, V.I., and Jones, J.D. (1997). AtDMC1, the Arabidopsis homologue of the yeast DMC1 gene: characterization, transposon-induced allelic variation and meiosis-associated expression. The Plant journal : for cell and molecular biology 11:1-14.


Li, L., Wu, W., Zhao, Y., and Zheng, B. (2017). A reciprocal inhibition between ARID1 and MET1 in male and female gametes inArabidopsis. Journal of integrative plant biology 59:657-668.


Li, X., Lei, M., Yan, Z., Wang, Q., Chen, A., Sun, J., Luo, D., and Wang, Y. (2014). The REL3-mediated TAS3 ta-siRNA pathway integratesauxin and ethylene signaling to regulate nodulation in Lotus japonicus. New Phytol 201:531-544.


Liu, X., Dinh, T.T., Li, D., Shi, B., Li, Y., Cao, X., Guo, L., Pan, Y., Jiao, Y., and Chen, X. (2014). AUXIN RESPONSE FACTOR 3 integratesthe functions of AGAMOUS and APETALA2 in floral meristem determinacy. The Plant journal : for cell and molecular biology 80:629-641.


Luo, Q.J., Mittal, A., Jia, F., and Rock, C.D. (2012). An autoregulatory feedback loop involving PAP1 and TAS4 in response to sugars inArabidopsis. Plant Mol Biol 80:117-129.


Marin, E., Jouannet, V., Herz, A., Lokerse, A.S., Weijers, D., Vaucheret, H., Nussaume, L., Crespi, M.D., and Maizel, A. (2010). miR390,Arabidopsis TAS3 tasiRNAs, and Their AUXIN RESPONSE FACTOR Targets Define an Autoregulatory Network QuantitativelyRegulating Lateral Root Growth. The Plant cell 22:1104-1117.


https://www.ncbi.nlm.nih.gov/pubmed?term=Garcia%2C D%2E%2C Collier%2C S%2EA%2E%2C Byrne%2C M%2EE%2E%2C and Martienssen%2C R%2EA%2E %282006%29%2E Specification of leaf polarity in Arabidopsis via the trans%2Dacting siRNA pathway%2E Current Biology&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Garcia,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Specification of leaf polarity in Arabidopsis via the trans-acting siRNA pathway&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Specification of leaf polarity in Arabidopsis via the trans-acting siRNA pathway&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Garcia,&as_ylo=2006&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Gasciolli%2C V%2E%2C Mallory%2C A%2EC%2E%2C Bartel%2C D%2EP%2E%2C and Vaucheret%2C H%2E %282005%29%2E Partially redundant functions of Arabidopsis DICER%2Dlike enzymes and a role for DCL4 in producing trans%2Dacting siRNAs%2E Current Biology&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Gasciolli,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Partially redundant functions of Arabidopsis DICER-like enzymes and a role for DCL4 in producing trans-acting siRNAs&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Partially redundant functions of Arabidopsis DICER-like enzymes and a role for DCL4 in producing trans-acting siRNAs&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Gasciolli,&as_ylo=2005&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Gross%2DHardt%2C R%2E%2C Lenhard%2C M%2E%2C and Laux%2C T%2E %282002%29%2E WUSCHEL signaling functions in interregional communication during Arabidopsis ovule development%2E Genes %26 development&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Gross-Hardt,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=WUSCHEL signaling functions in interregional communication during Arabidopsis ovule development&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=WUSCHEL signaling functions in interregional communication during Arabidopsis ovule development&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Gross-Hardt,&as_ylo=2002&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Grossniklaus%2C U%2E %282011%29%2E Plant germline development%3A a tale of cross%2Dtalk%2C signaling%2C and cellular interactions%2E Sex Plant Reprod&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Grossniklaus,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Plant germline development: a tale of cross-talk, signaling, and cellular interactions&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Plant germline development: a tale of cross-talk, signaling, and cellular interactions&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Grossniklaus,&as_ylo=2011&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Hejatko%2C J%2E%2C Blilou%2C I%2E%2C Brewer%2C P%2EB%2E%2C Friml%2C J%2E%2C Scheres%2C B%2E%2C and Benkova%2C E%2E %282006%29%2E In situ hybridization technique for mRNA detection in whole mount Arabidopsis samples%2E Nature protocols&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Hejatko,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=In situ hybridization technique for mRNA detection in whole mount Arabidopsis samples&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=In situ hybridization technique for mRNA detection in whole mount Arabidopsis samples&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Hejatko,&as_ylo=2006&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Hunter%2C C%2E%2C Willmann%2C M%2ER%2E%2C Wu%2C G%2E%2C Yoshikawa%2C M%2E%2C de la Luz Gutierrez%2DNava%2C M%2E%2C and Poethig%2C S%2ER%2E %282006%29%2E Trans%2Dacting siRNA%2Dmediated repression of ETTIN and ARF4 regulates heteroblasty in Arabidopsis%2E Development&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Hunter,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Trans-acting siRNA-mediated repression of ETTIN and ARF4 regulates heteroblasty in Arabidopsis&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Trans-acting siRNA-mediated repression of ETTIN and ARF4 regulates heteroblasty in Arabidopsis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Hunter,&as_ylo=2006&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Imran Siddiqi%2C G%2EG%2E%2C Ueli Grossniklaus%2C Veeraputhiran Subbiah%2E %282000%29%2E The dyad gene is required for progression through female meiosis in Arabidopsis%2E Development&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Imran&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=The dyad gene is required for progression through female meiosis in Arabidopsis.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=The dyad gene is required for progression through female meiosis in Arabidopsis.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Imran&as_ylo=2000&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Jauvion%2C V%2E%2C Elmayan%2C T%2E%2C and Vaucheret%2C H%2E %282010%29%2E The Conserved RNA Trafficking Proteins HPR1 and TEX1 Are Involved in the Production of Endogenous and Exogenous Small Interfering RNA in Arabidopsis%2E The Plant cell&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Jauvion,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=The Conserved RNA Trafficking Proteins HPR1 and TEX1 Are Involved in the Production of Endogenous and Exogenous Small Interfering RNA in Arabidopsis&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=The Conserved RNA Trafficking Proteins HPR1 and TEX1 Are Involved in the Production of Endogenous and Exogenous Small Interfering RNA in Arabidopsis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Jauvion,&as_ylo=2010&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Kidner%2C C%2E%2C and Timmermans%2C M%2E %282006%29%2E In situ hybridization as a tool to study the role of microRNAs in plant development%2E Methods in molecular biology&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Kidner,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=In situ hybridization as a tool to study the role of microRNAs in plant development&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=In situ hybridization as a tool to study the role of microRNAs in plant development&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Kidner,&as_ylo=2006&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Klimyuk%2C V%2EI%2E%2C and Jones%2C J%2ED%2E %281997%29%2E AtDMC1%2C the Arabidopsis homologue of the yeast DMC1 gene%3A characterization%2C transposon%2Dinduced allelic variation and meiosis%2Dassociated expression%2E The Plant journal %3A for cell and molecular biology&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Klimyuk,&hl=en&c2coff=1


https://scholar.google.com/scholar?as_q=&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Klimyuk,&as_ylo=1997&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Li%2C L%2E%2C Wu%2C W%2E%2C Zhao%2C Y%2E%2C and Zheng%2C B%2E %282017%29%2E A reciprocal inhibition between ARID1 and MET1 in male and female gametes in Arabidopsis%2E Journal of integrative plant biology&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Li,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=A reciprocal inhibition between ARID1 and MET1 in male and female gametes in Arabidopsis&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=A reciprocal inhibition between ARID1 and MET1 in male and female gametes in Arabidopsis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Li,&as_ylo=2017&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Li%2C X%2E%2C Lei%2C M%2E%2C Yan%2C Z%2E%2C Wang%2C Q%2E%2C Chen%2C A%2E%2C Sun%2C J%2E%2C Luo%2C D%2E%2C and Wang%2C Y%2E %282014%29%2E The REL3%2Dmediated TAS3 ta%2DsiRNA pathway integrates auxin and ethylene signaling to regulate nodulation in Lotus japonicus%2E New Phytol&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Li,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=The REL3-mediated TAS3 ta-siRNA pathway integrates auxin and ethylene signaling to regulate nodulation in Lotus japonicus&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=The REL3-mediated TAS3 ta-siRNA pathway integrates auxin and ethylene signaling to regulate nodulation in Lotus japonicus&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Li,&as_ylo=2014&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Liu%2C X%2E%2C Dinh%2C T%2ET%2E%2C Li%2C D%2E%2C Shi%2C B%2E%2C Li%2C Y%2E%2C Cao%2C X%2E%2C Guo%2C L%2E%2C Pan%2C Y%2E%2C Jiao%2C Y%2E%2C and Chen%2C X%2E %282014%29%2E AUXIN RESPONSE FACTOR 3 integrates the functions of AGAMOUS and APETALA2 in floral meristem determinacy%2E The Plant journal %3A for cell and molecular biology&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Liu,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=AUXIN RESPONSE FACTOR 3 integrates the functions of AGAMOUS and APETALA2 in floral meristem determinacy&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=AUXIN RESPONSE FACTOR 3 integrates the functions of AGAMOUS and APETALA2 in floral meristem determinacy&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Liu,&as_ylo=2014&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Luo%2C Q%2EJ%2E%2C Mittal%2C A%2E%2C Jia%2C F%2E%2C and Rock%2C C%2ED%2E %282012%29%2E An autoregulatory feedback loop involving PAP1 and TAS4 in response to sugars in Arabidopsis%2E Plant Mol Biol&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Luo,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=An autoregulatory feedback loop involving PAP1 and TAS4 in response to sugars in Arabidopsis&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=An autoregulatory feedback loop involving PAP1 and TAS4 in response to sugars in Arabidopsis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Luo,&as_ylo=2012&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Marin%2C E%2E%2C Jouannet%2C V%2E%2C Herz%2C A%2E%2C Lokerse%2C A%2ES%2E%2C Weijers%2C D%2E%2C Vaucheret%2C H%2E%2C Nussaume%2C L%2E%2C Crespi%2C M%2ED%2E%2C and Maizel%2C A%2E %282010%29%2E miR390%2C Arabidopsis TAS3 tasiRNAs%2C and Their AUXIN RESPONSE FACTOR Targets Define an Autoregulatory Network Quantitatively Regulating Lateral Root Growth%2E The Plant cell&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Marin,&hl=en&c2coff=1


https://scholar.google.com/scholar?as_q=&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Marin,&as_ylo=2010&as_allsubj=all&hl=en&c2coff=1

Mary C. Webb, B.E.S.G. (1990). Embryo sac development in Arabidopsis thaliana. Sex Plant Reprod 3:13.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Montgomery, T.A., Howell, M.D., Cuperus, J.T., Li, D., Hansen, J.E., Alexander, A.L., Chapman, E.J., Fahlgren, N., Allen, E., andCarrington, J.C. (2008a). Specificity of ARGONAUTE7-miR390 interaction and dual functionality in TAS3 trans-acting siRNA formation.Cell 133:128-141.


Montgomery, T.A., Yoo, S.J., Fahlgren, N., Gilbert, S.D., Howell, M.D., Sullivan, C.M., Alexander, A., Nguyen, G., Allen, E., Ahn, J.H., et al.(2008b). AGO1-miR173 complex initiates phased siRNA formation in plants. Proceedings of the National Academy of Sciences of theUnited States of America 105:20055-20062.


Nakagawa, T., Kurose, T., Hino, T., Tanaka, K., Kawamukai, M., Niwa, Y., Toyooka, K., Matsuoka, K., Jinbo, T., and Kimura, T. (2007).Development of series of gateway binary vectors, pGWBs, for realizing efficient construction of fusion genes for plant transformation.J Biosci Bioeng 104:34-41.


Nogueira, F.T., Madi, S., Chitwood, D.H., Juarez, M.T., and Timmermans, M.C. (2007). Two small regulatory RNAs establish opposingfates of a developmental axis. Genes & development 21:750-755.


Oh, S.A., Pal, M.D., Park, S.K., Johnson, J.A., and Twell, D. (2010). The tobacco MAP215/Dis1-family protein TMBP200 is required for thefunctional organization of microtubule arrays during male germline establishment. J Exp Bot 61:969-981.


Olmedo-Monfil, V., Duran-Figueroa, N., Arteaga-Vazquez, M., Demesa-Arevalo, E., Autran, D., Grimanelli, D., Slotkin, R.K., Martienssen,R.A., and Vielle-Calzada, J.P. (2010). Control of female gamete formation by a small RNA pathway in Arabidopsis. Nature 464:628-632.


Ozerova, L.V., Krasnikova, M.S., Troitsky, A.V., Solovyev, A.G., and Morozov, S.Y. (2013). TAS3 Genes for small ta-siARF RNAs in plantsbelonging to subtribe Senecioninae: occurrence of prematurely terminated RNA precursors. Mol Gen Mikrobiol Virusol:33-36.


Payne, T., Johnson, S.D., and Koltunow, A.M. (2004). KNUCKLES (KNU) encodes a C2H2 zinc-finger protein that regulatesdevelopment of basal pattern elements of the Arabidopsis gynoecium. Development 131:3737-3749.


Peragine, A., Yoshikawa, M., Wu, G., Albrecht, H.L., and Poethig, R.S. (2004). SGS3 and SGS2/SDE1/RDR6 are required for juveniledevelopment and the production of trans-acting siRNAs in Arabidopsis. Genes & development 18:2368-2379.


Rajagopalan, R., Vaucheret, H., Trejo, J., and Bartel, D.P. (2006). A diverse and evolutionarily fluid set of microRNAs in Arabidopsisthaliana. Genes & development 20:3407-3425.


Robinson-Beers, K., Pruitt, R.E., and Gasser, C.S. (1992). Ovule Development in Wild-Type Arabidopsis and Two Female-SterileMutants. The Plant cell 4:1237-1249.


Rodriguez-Leal, D., Leon-Martinez, G., Abad-Vivero, U., and Vielle-Calzada, J.P. (2015). Natural variation in epigenetic pathways affectsthe specification of female gamete precursors in Arabidopsis. The Plant cell 27:1034-1045.


Schiefthaler, U., Balasubramanian, S., Sieber, P., Chevalier, D., Wisman, E., and Schneitz, K. (1999). Molecular analysis of NOZZLE, agene involved in pattern formation and early sporogenesis during sex organ development in Arabidopsis thaliana. Proceedings of theNational Academy of Sciences of the United States of America 96:11664-11669.


Schmidt, A., Wuest, S.E., Vijverberg, K., Baroux, C., Kleen, D., and Grossniklaus, U. (2011). Transcriptome analysis of the Arabidopsis

https://www.ncbi.nlm.nih.gov/pubmed?term=Mary C%2E Webb%2C B%2EE%2ES%2EG%2E %281990%29%2E Embryo sac development in Arabidopsis thaliana%2E Sex Plant Reprod&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Mary&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Embryo sac development in Arabidopsis thaliana&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Embryo sac development in Arabidopsis thaliana&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Mary&as_ylo=1990&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Montgomery%2C T%2EA%2E%2C Howell%2C M%2ED%2E%2C Cuperus%2C J%2ET%2E%2C Li%2C D%2E%2C Hansen%2C J%2EE%2E%2C Alexander%2C A%2EL%2E%2C Chapman%2C E%2EJ%2E%2C Fahlgren%2C N%2E%2C Allen%2C E%2E%2C and Carrington%2C J%2EC%2E %282008a%29%2E Specificity of ARGONAUTE7%2DmiR390 interaction and dual functionality in TAS3 trans%2Dacting siRNA formation%2E Cell&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Montgomery,&hl=en&c2coff=1


https://scholar.google.com/scholar?as_q=&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Montgomery,&as_ylo=2008&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Montgomery%2C T%2EA%2E%2C Yoo%2C S%2EJ%2E%2C Fahlgren%2C N%2E%2C Gilbert%2C S%2ED%2E%2C Howell%2C M%2ED%2E%2C Sullivan%2C C%2EM%2E%2C Alexander%2C A%2E%2C Nguyen%2C G%2E%2C Allen%2C E%2E%2C Ahn%2C J%2EH%2E%2C et al%2E %282008b%29%2E AGO1%2DmiR173 complex initiates phased siRNA formation in plants%2E Proceedings of the National Academy of Sciences of the United States of America&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Montgomery,&hl=en&c2coff=1


https://scholar.google.com/scholar?as_q=&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Montgomery,&as_ylo=2008&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Nakagawa%2C T%2E%2C Kurose%2C T%2E%2C Hino%2C T%2E%2C Tanaka%2C K%2E%2C Kawamukai%2C M%2E%2C Niwa%2C Y%2E%2C Toyooka%2C K%2E%2C Matsuoka%2C K%2E%2C Jinbo%2C T%2E%2C and Kimura%2C T%2E %282007%29%2E Development of series of gateway binary vectors%2C pGWBs%2C for realizing efficient construction of fusion genes for plant transformation%2E J Biosci Bioeng&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Nakagawa,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Development of series of gateway binary vectors, pGWBs, for realizing efficient construction of fusion genes for plant transformation&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Development of series of gateway binary vectors, pGWBs, for realizing efficient construction of fusion genes for plant transformation&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Nakagawa,&as_ylo=2007&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Nogueira%2C F%2ET%2E%2C Madi%2C S%2E%2C Chitwood%2C D%2EH%2E%2C Juarez%2C M%2ET%2E%2C and Timmermans%2C M%2EC%2E %282007%29%2E Two small regulatory RNAs establish opposing fates of a developmental axis%2E Genes %26 development&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Nogueira,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Two small regulatory RNAs establish opposing fates of a developmental axis&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Two small regulatory RNAs establish opposing fates of a developmental axis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Nogueira,&as_ylo=2007&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Oh%2C S%2EA%2E%2C Pal%2C M%2ED%2E%2C Park%2C S%2EK%2E%2C Johnson%2C J%2EA%2E%2C and Twell%2C D%2E %282010%29%2E The tobacco MAP215%2FDis1%2Dfamily protein TMBP200 is required for the functional organization of microtubule arrays during male germline establishment%2E J Exp Bot&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Oh,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=The tobacco MAP215/Dis1-family protein TMBP200 is required for the functional organization of microtubule arrays during male germline establishment&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=The tobacco MAP215/Dis1-family protein TMBP200 is required for the functional organization of microtubule arrays during male germline establishment&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Oh,&as_ylo=2010&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Olmedo%2DMonfil%2C V%2E%2C Duran%2DFigueroa%2C N%2E%2C Arteaga%2DVazquez%2C M%2E%2C Demesa%2DArevalo%2C E%2E%2C Autran%2C D%2E%2C Grimanelli%2C D%2E%2C Slotkin%2C R%2EK%2E%2C Martienssen%2C R%2EA%2E%2C and Vielle%2DCalzada%2C J%2EP%2E %282010%29%2E Control of female gamete formation by a small RNA pathway in Arabidopsis%2E Nature&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Olmedo-Monfil,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Control of female gamete formation by a small RNA pathway in Arabidopsis&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Control of female gamete formation by a small RNA pathway in Arabidopsis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Olmedo-Monfil,&as_ylo=2010&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Ozerova%2C L%2EV%2E%2C Krasnikova%2C M%2ES%2E%2C Troitsky%2C A%2EV%2E%2C Solovyev%2C A%2EG%2E%2C and Morozov%2C S%2EY%2E %282013%29%2E TAS3 Genes for small ta%2DsiARF RNAs in plants belonging to subtribe Senecioninae%3A occurrence of prematurely terminated RNA precursors%2E Mol Gen Mikrobiol Virusol&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Ozerova,&hl=en&c2coff=1


https://scholar.google.com/scholar?as_q=&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Ozerova,&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Payne%2C T%2E%2C Johnson%2C S%2ED%2E%2C and Koltunow%2C A%2EM%2E %282004%29%2E KNUCKLES %28KNU%29 encodes a C2H2 zinc%2Dfinger protein that regulates development of basal pattern elements of the Arabidopsis gynoecium%2E Development&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Payne,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=KNUCKLES (KNU) encodes a C2H2 zinc-finger protein that regulates development of basal pattern elements of the Arabidopsis gynoecium&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=KNUCKLES (KNU) encodes a C2H2 zinc-finger protein that regulates development of basal pattern elements of the Arabidopsis gynoecium&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Payne,&as_ylo=2004&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Peragine%2C A%2E%2C Yoshikawa%2C M%2E%2C Wu%2C G%2E%2C Albrecht%2C H%2EL%2E%2C and Poethig%2C R%2ES%2E %282004%29%2E SGS3 and SGS2%2FSDE1%2FRDR6 are required for juvenile development and the production of trans%2Dacting siRNAs in Arabidopsis%2E Genes %26 development&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Peragine,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=SGS3 and SGS2/SDE1/RDR6 are required for juvenile development and the production of trans-acting siRNAs in Arabidopsis&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=SGS3 and SGS2/SDE1/RDR6 are required for juvenile development and the production of trans-acting siRNAs in Arabidopsis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Peragine,&as_ylo=2004&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Rajagopalan%2C R%2E%2C Vaucheret%2C H%2E%2C Trejo%2C J%2E%2C and Bartel%2C D%2EP%2E %282006%29%2E A diverse and evolutionarily fluid set of microRNAs in Arabidopsis thaliana%2E Genes %26 development&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Rajagopalan,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=A diverse and evolutionarily fluid set of microRNAs in Arabidopsis thaliana&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=A diverse and evolutionarily fluid set of microRNAs in Arabidopsis thaliana&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Rajagopalan,&as_ylo=2006&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Robinson%2DBeers%2C K%2E%2C Pruitt%2C R%2EE%2E%2C and Gasser%2C C%2ES%2E %281992%29%2E Ovule Development in Wild%2DType Arabidopsis and Two Female%2DSterile Mutants%2E The Plant cell&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Robinson-Beers,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Ovule Development in Wild-Type Arabidopsis and Two Female-Sterile Mutants&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Ovule Development in Wild-Type Arabidopsis and Two Female-Sterile Mutants&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Robinson-Beers,&as_ylo=1992&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Rodriguez%2DLeal%2C D%2E%2C Leon%2DMartinez%2C G%2E%2C Abad%2DVivero%2C U%2E%2C and Vielle%2DCalzada%2C J%2EP%2E %282015%29%2E Natural variation in epigenetic pathways affects the specification of female gamete precursors in Arabidopsis%2E The Plant cell&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Rodriguez-Leal,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Natural variation in epigenetic pathways affects the specification of female gamete precursors in Arabidopsis&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Natural variation in epigenetic pathways affects the specification of female gamete precursors in Arabidopsis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Rodriguez-Leal,&as_ylo=2015&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Schiefthaler%2C U%2E%2C Balasubramanian%2C S%2E%2C Sieber%2C P%2E%2C Chevalier%2C D%2E%2C Wisman%2C E%2E%2C and Schneitz%2C K%2E %281999%29%2E Molecular analysis of NOZZLE%2C a gene involved in pattern formation and early sporogenesis during sex organ development in Arabidopsis thaliana%2E Proceedings of the National Academy of Sciences of the United States of America&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Schiefthaler,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Molecular analysis of NOZZLE, a gene involved in pattern formation and early sporogenesis during sex organ development in Arabidopsis thaliana&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Molecular analysis of NOZZLE, a gene involved in pattern formation and early sporogenesis during sex organ development in Arabidopsis thaliana&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Schiefthaler,&as_ylo=1999&as_allsubj=all&hl=en&c2coff=1

megaspore mother cell uncovers the importance of RNA helicases for plant germline development. PLoS biology 9:e1001155.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Shirley, N.J., Aubert, M.K., Wilkinson, L.G., Bird, D.C., Lora, J., Yang, X.J., and Tucker, M.R. (2019). Translating auxin responses intoovules, seeds and yield: Insight from Arabidopsis and the cereals. Journal of integrative plant biology 61:310-336.


Sieber, P., Gheyselinck, J., Gross-Hardt, R., Laux, T., Grossniklaus, U., and Schneitz, K. (2004). Pattern formation during early ovuledevelopment in Arabidopsis thaliana. Dev Biol 273:321-334.


Simonini, S., Bencivenga, S., Trick, M., and Ostergaard, L. (2017). Auxin-Induced Modulation of ETTIN Activity Orchestrates GeneExpression in Arabidopsis. The Plant cell 29:1864-1882.


Singh, M., Goel, S., Meeley, R.B., Dantec, C., Parrinello, H., Michaud, C., Leblanc, O., and Grimanelli, D. (2011). Production of viablegametes without meiosis in maize deficient for an ARGONAUTE protein. The Plant cell 23:443-458.


Singh, S.K., Kumar, V., Srinivasan, R., Ahuja, P.S., Bhat, S.R., and Sreenivasulu, Y. (2017). The TRAF Mediated GametogenesisProgression (TRAMGaP) Gene Is Required for Megaspore Mother Cell Specification and Gametophyte Development. Plant physiology175:1220-1237.


Su, Z., Zhao, L., Zhao, Y., Li, S., Won, S., Cai, H., Wang, L., Li, Z., Chen, P., Qin, Y., et al. (2017). The THO Complex Non-Cell-Autonomously Represses Female Germline Specification through the TAS3-ARF3 Module. Current biology : CB 27:1597-1609 e1592.


Vazquez, F., Vaucheret, H., Rajagopalan, R., Lepers, C., Gasciolli, V., Mallory, A.C., Hilbert, J.L., Bartel, D.P., and Crete, P. (2004).Endogenous trans-acting siRNAs regulate the accumulation of Arabidopsis mRNAs. Molecular cell 16:69-79.


Walbot, V., and Evans, M.M. (2003). Unique features of the plant life cycle and their consequences. Nat Rev Genet 4:369-379.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Wang, J., Gao, X., Li, L., Shi, X., Zhang, J., and Shi, Z. (2010). Overexpression of Osta-siR2141 caused abnormal polarity establishmentand retarded growth in rice. J Exp Bot 61:1885-1895.


Wei, B., Zhang, J., Pang, C., Yu, H., Guo, D., Jiang, H., Ding, M., Chen, Z., Tao, Q., Gu, H., et al. (2015). The molecular mechanism ofsporocyteless/nozzle in controlling Arabidopsis ovule development. Cell research 25:121-134.


Williams, L., Carles, C.C., Osmont, K.S., and Fletcher, J.C. (2005). A database analysis method identifies an endogenous trans-actingshort-interfering RNA that targets the Arabidopsis ARF2, ARF3, and ARF4 genes. Proceedings of the National Academy of Sciences ofthe United States of America 102:9703-9708.


Xia, R., Xu, J., and Meyers, B.C. (2017). The Emergence, Evolution, and Diversification of the miR390-TAS3-ARF Pathway in LandPlants. The Plant cell 29:1232-1247.


Xie, Z.X., Allen, E., Wilken, A., and Carrington, J.C. (2005). DICER-LIKE 4 functions in trans-acting small interfering RNA biogenesis andvegetative phase change in Arabidopsis thaliana. Proceedings of the National Academy of Sciences of the United States of America102:12984-12989.


Yang, W.C., Shi, D.Q., and Chen, Y.H. (2010). Female gametophyte development in flowering plants. Annual review of plant biology61:89-108.


https://www.ncbi.nlm.nih.gov/pubmed?term=Schmidt%2C A%2E%2C Wuest%2C S%2EE%2E%2C Vijverberg%2C K%2E%2C Baroux%2C C%2E%2C Kleen%2C D%2E%2C and Grossniklaus%2C U%2E %282011%29%2E Transcriptome analysis of the Arabidopsis megaspore mother cell uncovers the importance of RNA helicases for plant germline development%2E PLoS biology 9%3Ae&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Schmidt,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Transcriptome analysis of the Arabidopsis megaspore mother cell uncovers the importance of RNA helicases for plant germline development&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Transcriptome analysis of the Arabidopsis megaspore mother cell uncovers the importance of RNA helicases for plant germline development&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Schmidt,&as_ylo=2011&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Shirley%2C N%2EJ%2E%2C Aubert%2C M%2EK%2E%2C Wilkinson%2C L%2EG%2E%2C Bird%2C D%2EC%2E%2C Lora%2C J%2E%2C Yang%2C X%2EJ%2E%2C and Tucker%2C M%2ER%2E %282019%29%2E Translating auxin responses into ovules%2C seeds and yield%3A Insight from Arabidopsis and the cereals%2E Journal of integrative plant biology&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Shirley,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Translating auxin responses into ovules, seeds and yield: Insight from Arabidopsis and the cereals&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Translating auxin responses into ovules, seeds and yield: Insight from Arabidopsis and the cereals&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Shirley,&as_ylo=2019&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Sieber%2C P%2E%2C Gheyselinck%2C J%2E%2C Gross%2DHardt%2C R%2E%2C Laux%2C T%2E%2C Grossniklaus%2C U%2E%2C and Schneitz%2C K%2E %282004%29%2E Pattern formation during early ovule development in Arabidopsis thaliana%2E Dev Biol&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Sieber,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Pattern formation during early ovule development in Arabidopsis thaliana&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Pattern formation during early ovule development in Arabidopsis thaliana&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Sieber,&as_ylo=2004&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Simonini%2C S%2E%2C Bencivenga%2C S%2E%2C Trick%2C M%2E%2C and Ostergaard%2C L%2E %282017%29%2E Auxin%2DInduced Modulation of ETTIN Activity Orchestrates Gene Expression in Arabidopsis%2E The Plant cell&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Simonini,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Auxin-Induced Modulation of ETTIN Activity Orchestrates Gene Expression in Arabidopsis&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Auxin-Induced Modulation of ETTIN Activity Orchestrates Gene Expression in Arabidopsis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Simonini,&as_ylo=2017&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Singh%2C M%2E%2C Goel%2C S%2E%2C Meeley%2C R%2EB%2E%2C Dantec%2C C%2E%2C Parrinello%2C H%2E%2C Michaud%2C C%2E%2C Leblanc%2C O%2E%2C and Grimanelli%2C D%2E %282011%29%2E Production of viable gametes without meiosis in maize deficient for an ARGONAUTE protein%2E The Plant cell&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Singh,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Production of viable gametes without meiosis in maize deficient for an ARGONAUTE protein&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Production of viable gametes without meiosis in maize deficient for an ARGONAUTE protein&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Singh,&as_ylo=2011&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Singh%2C S%2EK%2E%2C Kumar%2C V%2E%2C Srinivasan%2C R%2E%2C Ahuja%2C P%2ES%2E%2C Bhat%2C S%2ER%2E%2C and Sreenivasulu%2C Y%2E %282017%29%2E The TRAF Mediated Gametogenesis Progression %28TRAMGaP%29 Gene Is Required for Megaspore Mother Cell Specification and Gametophyte Development%2E Plant physiology&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Singh,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=The TRAF Mediated Gametogenesis Progression (TRAMGaP) Gene Is Required for Megaspore Mother Cell Specification and Gametophyte Development&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=The TRAF Mediated Gametogenesis Progression (TRAMGaP) Gene Is Required for Megaspore Mother Cell Specification and Gametophyte Development&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Singh,&as_ylo=2017&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Su%2C Z%2E%2C Zhao%2C L%2E%2C Zhao%2C Y%2E%2C Li%2C S%2E%2C Won%2C S%2E%2C Cai%2C H%2E%2C Wang%2C L%2E%2C Li%2C Z%2E%2C Chen%2C P%2E%2C Qin%2C Y%2E%2C et al%2E %282017%29%2E The THO Complex Non%2DCell%2DAutonomously Represses Female Germline Specification through the TAS3%2DARF3 Module%2E Current biology %3A CB 27%3A1597%2D1609 e&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Su,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=The THO Complex Non-Cell-Autonomously Represses Female Germline Specification through the TAS3-ARF3 Module&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=The THO Complex Non-Cell-Autonomously Represses Female Germline Specification through the TAS3-ARF3 Module&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Su,&as_ylo=2017&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Vazquez%2C F%2E%2C Vaucheret%2C H%2E%2C Rajagopalan%2C R%2E%2C Lepers%2C C%2E%2C Gasciolli%2C V%2E%2C Mallory%2C A%2EC%2E%2C Hilbert%2C J%2EL%2E%2C Bartel%2C D%2EP%2E%2C and Crete%2C P%2E %282004%29%2E Endogenous trans%2Dacting siRNAs regulate the accumulation of Arabidopsis mRNAs%2E Molecular cell&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Vazquez,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Endogenous trans-acting siRNAs regulate the accumulation of Arabidopsis mRNAs&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Endogenous trans-acting siRNAs regulate the accumulation of Arabidopsis mRNAs&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Vazquez,&as_ylo=2004&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Walbot%2C V%2E%2C and Evans%2C M%2EM%2E %282003%29%2E Unique features of the plant life cycle and their consequences%2E Nat Rev Genet&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Walbot,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Unique features of the plant life cycle and their consequences&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Unique features of the plant life cycle and their consequences&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Walbot,&as_ylo=2003&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Wang%2C J%2E%2C Gao%2C X%2E%2C Li%2C L%2E%2C Shi%2C X%2E%2C Zhang%2C J%2E%2C and Shi%2C Z%2E %282010%29%2E Overexpression of Osta%2DsiR2141 caused abnormal polarity establishment and retarded growth in rice%2E J Exp Bot&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Wang,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Overexpression of Osta-siR2141 caused abnormal polarity establishment and retarded growth in rice&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Overexpression of Osta-siR2141 caused abnormal polarity establishment and retarded growth in rice&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Wang,&as_ylo=2010&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Wei%2C B%2E%2C Zhang%2C J%2E%2C Pang%2C C%2E%2C Yu%2C H%2E%2C Guo%2C D%2E%2C Jiang%2C H%2E%2C Ding%2C M%2E%2C Chen%2C Z%2E%2C Tao%2C Q%2E%2C Gu%2C H%2E%2C et al%2E %282015%29%2E The molecular mechanism of sporocyteless%2Fnozzle in controlling Arabidopsis ovule development%2E Cell research&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Wei,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=The molecular mechanism of sporocyteless/nozzle in controlling Arabidopsis ovule development&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=The molecular mechanism of sporocyteless/nozzle in controlling Arabidopsis ovule development&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Wei,&as_ylo=2015&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Williams%2C L%2E%2C Carles%2C C%2EC%2E%2C Osmont%2C K%2ES%2E%2C and Fletcher%2C J%2EC%2E %282005%29%2E A database analysis method identifies an endogenous trans%2Dacting short%2Dinterfering RNA that targets the Arabidopsis ARF2%2C ARF3%2C and ARF4 genes%2E Proceedings of the National Academy of Sciences of the United States of America&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Williams,&hl=en&c2coff=1


https://scholar.google.com/scholar?as_q=&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Williams,&as_ylo=2005&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Xia%2C R%2E%2C Xu%2C J%2E%2C and Meyers%2C B%2EC%2E %282017%29%2E The Emergence%2C Evolution%2C and Diversification of the miR390%2DTAS3%2DARF Pathway in Land Plants%2E The Plant cell&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Xia,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=The Emergence, Evolution, and Diversification of the miR390-TAS3-ARF Pathway in Land Plants&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=The Emergence, Evolution, and Diversification of the miR390-TAS3-ARF Pathway in Land Plants&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Xia,&as_ylo=2017&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Xie%2C Z%2EX%2E%2C Allen%2C E%2E%2C Wilken%2C A%2E%2C and Carrington%2C J%2EC%2E %282005%29%2E DICER%2DLIKE 4 functions in trans%2Dacting small interfering RNA biogenesis and vegetative phase change in Arabidopsis thaliana%2E Proceedings of the National Academy of Sciences of the United States of America&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Xie,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=DICER-LIKE 4 functions in trans-acting small interfering RNA biogenesis and vegetative phase change in Arabidopsis thaliana&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=DICER-LIKE 4 functions in trans-acting small interfering RNA biogenesis and vegetative phase change in Arabidopsis thaliana&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Xie,&as_ylo=2005&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Yang%2C W%2EC%2E%2C Shi%2C D%2EQ%2E%2C and Chen%2C Y%2EH%2E %282010%29%2E Female gametophyte development in flowering plants%2E Annual review of plant biology&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Yang,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Female gametophyte development in flowering plants&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Female gametophyte development in flowering plants&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Yang,&as_ylo=2010&as_allsubj=all&hl=en&c2coff=1

Google Scholar: Author Only Title Only Author and Title

Yang, W.C., Ye, D., Xu, J., and Sundaresan, V. (1999). The SPOROCYTELESS gene of Arabidopsis is required for initiation ofsporogenesis and encodes a novel nuclear protein. Genes & development 13:2108-2117.


Yao, X., Yang, H., Zhu, Y., Xue, J., Wang, T., Song, T., Yang, Z., and Wang, S. (2018). The Canonical E2Fs Are Required for GermlineDevelopment in Arabidopsis. Frontiers in plant science 9:638.


Yelina, N.E., Smith, L.M., Jones, A.M., Patel, K., Kelly, K.A., and Baulcombe, D.C. (2010). Putative Arabidopsis THO/TREX mRNA exportcomplex is involved in transgene and endogenous siRNA biosynthesis. Proceedings of the National Academy of Sciences of theUnited States of America 107:13948-13953.


Yoshikawa, M., Iki, T., Tsutsui, Y., Miyashita, K., Poethig, R.S., Habu, Y., and Ishikawa, M. (2013). 3' fragment of miR173-programmedRISC-cleaved RNA is protected from degradation in a complex with RISC and SGS3. Proceedings of the National Academy of Sciencesof the United States of America 110:4117-4122.


Yoshikawa, M., Peragine, A., Park, M.Y., and Poethig, R.S. (2005). A pathway for the biogenesis of trans-acting siRNAs in Arabidopsis.Genes & development 19:2164-2175.


Zhao, L., Cai, H., Su, Z., Wang, L., Huang, X., Zhang, M., Chen, P., Dai, X., Zhao, H., Palanivelu, R., et al. (2018). KLU suppressesmegasporocyte cell fate through SWR1-mediated activation of WRKY28 expression in Arabidopsis. Proceedings of the NationalAcademy of Sciences of the United States of America 115:E526-E535.


Zheng, Y., Zhang, K., Guo, L., Liu, X., and Zhang, Z. (2018). AUXIN RESPONSE FACTOR3 plays distinct role during early flowerdevelopment. Plant Signal Behav 13:e1467690.


https://www.ncbi.nlm.nih.gov/pubmed?term=Yang%2C W%2EC%2E%2C Ye%2C D%2E%2C Xu%2C J%2E%2C and Sundaresan%2C V%2E %281999%29%2E The SPOROCYTELESS gene of Arabidopsis is required for initiation of sporogenesis and encodes a novel nuclear protein%2E Genes %26 development&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Yang,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=The SPOROCYTELESS gene of Arabidopsis is required for initiation of sporogenesis and encodes a novel nuclear protein&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=The SPOROCYTELESS gene of Arabidopsis is required for initiation of sporogenesis and encodes a novel nuclear protein&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Yang,&as_ylo=1999&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Yao%2C X%2E%2C Yang%2C H%2E%2C Zhu%2C Y%2E%2C Xue%2C J%2E%2C Wang%2C T%2E%2C Song%2C T%2E%2C Yang%2C Z%2E%2C and Wang%2C S%2E %282018%29%2E The Canonical E2Fs Are Required for Germline Development in Arabidopsis%2E Frontiers in plant science&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Yao,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=The Canonical E2Fs Are Required for Germline Development in Arabidopsis&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=The Canonical E2Fs Are Required for Germline Development in Arabidopsis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Yao,&as_ylo=2018&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Yelina%2C N%2EE%2E%2C Smith%2C L%2EM%2E%2C Jones%2C A%2EM%2E%2C Patel%2C K%2E%2C Kelly%2C K%2EA%2E%2C and Baulcombe%2C D%2EC%2E %282010%29%2E Putative Arabidopsis THO%2FTREX mRNA export complex is involved in transgene and endogenous siRNA biosynthesis%2E Proceedings of the National Academy of Sciences of the United States of America&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Yelina,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Putative Arabidopsis THO/TREX mRNA export complex is involved in transgene and endogenous siRNA biosynthesis&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Putative Arabidopsis THO/TREX mRNA export complex is involved in transgene and endogenous siRNA biosynthesis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Yelina,&as_ylo=2010&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Yoshikawa%2C M%2E%2C Iki%2C T%2E%2C Tsutsui%2C Y%2E%2C Miyashita%2C K%2E%2C Poethig%2C R%2ES%2E%2C Habu%2C Y%2E%2C and Ishikawa%2C M%2E %282013%29%2E 3%27 fragment of miR173%2Dprogrammed RISC%2Dcleaved RNA is protected from degradation in a complex with RISC and SGS3%2E Proceedings of the National Academy of Sciences of the United States of America&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Yoshikawa,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=3' fragment of miR173-programmed RISC-cleaved RNA is protected from degradation in a complex with RISC and SGS3&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=3' fragment of miR173-programmed RISC-cleaved RNA is protected from degradation in a complex with RISC and SGS3&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Yoshikawa,&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Yoshikawa%2C M%2E%2C Peragine%2C A%2E%2C Park%2C M%2EY%2E%2C and Poethig%2C R%2ES%2E %282005%29%2E A pathway for the biogenesis of trans%2Dacting siRNAs in Arabidopsis%2E Genes %26 development&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Yoshikawa,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=A pathway for the biogenesis of trans-acting siRNAs in Arabidopsis&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=A pathway for the biogenesis of trans-acting siRNAs in Arabidopsis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Yoshikawa,&as_ylo=2005&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Zhao%2C L%2E%2C Cai%2C H%2E%2C Su%2C Z%2E%2C Wang%2C L%2E%2C Huang%2C X%2E%2C Zhang%2C M%2E%2C Chen%2C P%2E%2C Dai%2C X%2E%2C Zhao%2C H%2E%2C Palanivelu%2C R%2E%2C et al%2E %282018%29%2E KLU suppresses megasporocyte cell fate through SWR1%2Dmediated activation of WRKY28 expression in Arabidopsis%2E Proceedings of the National Academy of Sciences of the United States of America 115%3AE526%2DE&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Zhao,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=KLU suppresses megasporocyte cell fate through SWR1-mediated activation of WRKY28 expression in Arabidopsis&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=KLU suppresses megasporocyte cell fate through SWR1-mediated activation of WRKY28 expression in Arabidopsis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Zhao,&as_ylo=2018&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Zheng%2C Y%2E%2C Zhang%2C K%2E%2C Guo%2C L%2E%2C Liu%2C X%2E%2C and Zhang%2C Z%2E %282018%29%2E AUXIN RESPONSE FACTOR3 plays distinct role during early flower development%2E Plant Signal Behav 13%3Ae&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Zheng,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=AUXIN RESPONSE FACTOR3 plays distinct role during early flower development&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=AUXIN RESPONSE FACTOR3 plays distinct role during early flower development&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Zheng,&as_ylo=2018&as_allsubj=all&hl=en&c2coff=1

DOI 10.1105/tpc.20.00126; originally published online July 23, 2020;Plant Cell

Qin and Xuemei ChenZhenxia Su, Nannan Wang, Zhimin Hou, Baiyang Li, Dingning Li, Yanhui Liu, Hanyang Cai, Yuan

Regulation of Female Germline Specification via Small RNA Mobility in Arabidopsis

This information is current as of December 29, 2020

Supplemental Data /content/suppl/2020/07/23/tpc.20.00126.DC1.html

Permissions X

https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw1532298

eTOCs http://www.plantcell.org/cgi/alerts/ctmain

Sign up for eTOCs at:

CiteTrack Alerts http://www.plantcell.org/cgi/alerts/ctmain

Sign up for CiteTrack Alerts at:

Subscription Information http://www.aspb.org/publications/subscriptions.cfm

is available at:Plant Physiology and The Plant CellSubscription Information for

ADVANCING THE SCIENCE OF PLANT BIOLOGY © American Society of Plant Biologists

https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw1532298X

https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw1532298X

http://www.plantcell.org/cgi/alerts/ctmain

http://www.plantcell.org/cgi/alerts/ctmain

http://www.aspb.org/publications/subscriptions.cfm

regulation of female germline specification via small rna … · 2020. 7. 23. · germline...

Documents