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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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
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