1
Running Head: 1
Transcriptomic basis of a rice reproductive barrier 2
3
Corresponding Author: 4
Qifa Zhang 5
National Key Laboratory of Crop Genetic Improvement and National Center of Plant 6
Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China 7
Phone: 86-27-87282429 8
Fax: 86-27-87287092 9
E-mail: [email protected] 10
11
Research Area: 12
Genes, Development and Evolution 13
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Plant Physiology Preview. Published on May 8, 2017, as DOI:10.1104/pp.17.00093
Copyright 2017 by the American Society of Plant Biologists
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Title of article: 15
Reproductive barriers in indica-japonica rice hybrids are uncovered by transcriptome 16
analysis 17
All authors’ full names: 18
Yanfen Zhu, Yiming Yu, Ke Cheng, Yidan Ouyang, Jia Wang, Liang Gong, Qinghua 19
Zhang, Xianghua Li, Jinghua Xiao and Qifa Zhang* 20
*For correspondence (Fax: 86-27-87287092; Phone: 86-27-87282429; e-mail: 21
23
Author contributions: 24
Y.Z. and Qifa Zhang designed the research; Y.Z., Y.Y., K.C., Y.O., J.W., L.G. and 25
Qinghua Zhang performed the research; X.L. and J.X. contributed reagents; Y.Z. and 26
Qifa Zhang analyzed data and wrote the paper. 27
The authors declare no conflict of interests. 28
29
Affiliations: 30
National Key Laboratory of Crop Genetic Improvement and National Center of Plant 31
Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China 32
33
One Sentence Summary: 34
A reproductive barrier causes female gamete abortion of indica-japonica rice hybrids 35
by damaging cell wall integrity inducing massive biotic and abiotic stresses resulting 36
in ER stress leading to premature PCD. 37
38
Footnotes: 39
Financial source: This work was supported by grants from the National Key Research 40
and Development Program (2016YFD0100903) and National Natural Science 41
Foundation (31130032) of China. 42
43
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ABSTRACT 44
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In rice (Oryza sativa L.), hybrids between indica and japonica subspecies are usually 46
highly sterile, which provides a model system for studying postzygotic reproductive 47
isolation. A killer-protector system S5 composed of three adjacent genes, ORF3, 48
ORF4 and ORF5, regulates female gamete fertility of indica-japonica hybrids. To 49
characterize the processes underlying this system, we performed transcriptomic 50
analyses of pistils from rice variety Balilla (BL), Balilla with transformed ORF5+ 51
(BL5+) producing sterile female gametes, and Balilla with transformed ORF3+ and 52
ORF5+ (BL3+5+) producing fertile gametes. RNA sequencing of tissues collected 53
before (MMC), during (MEI), and after (AME) meiosis of the megaspore mother cell 54
detected 19,269 to 20,928 genes as expressed. Comparison between BL5+ and BL 55
showed that ORF5+ induced differential expression of 8,339, 6,278 and 530 genes at 56
MMC, MEI, and AME. At MMC, large-scale differential expression of cell wall 57
modifying genes and biotic and abiotic response genes indicated that cell wall 58
integrity damage induced severe biotic and abiotic stresses. The processes continued 59
to MEI and induced ER stress as indicated by differential expression of ER stress 60
responsive genes, leading to PCD at MEI and AME, resulting in abortive female 61
gametes. In the BL3+5+/BL comparison, 3,986, 749 and 370 genes were 62
differentially expressed at MMC, MEI and AME. Large numbers of cell wall 63
modification and biotic and abiotic response genes were also induced at MMC but 64
largely suppressed at MEI without inducing ER stress and PCD, producing fertile 65
gametes. These results have general implications for the understanding of biological 66
processes underlying reproductive barriers. 67
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INTRODUCTION 69
Reproductive isolation, a phenomenon widely existing in natural organisms, 70
promotes speciation and maintains the integrity of species over time (Coyne and Orr, 71
2004). It is divided into two general categories depending on whether it occurs before 72
or after fertilization: prezygotic reproductive isolation and postzygotic reproductive 73
isolation (Seehausen et al., 2014). The former prevents the formation of hybrid 74
zygotes, while the latter results in hybrid incompatibility including hybrid necrosis, 75
weakness, sterility, and lethality in F1 or later generations (Ouyang and Zhang, 2013). 76
The Dobzhansky-Muller model suggests that hybrid incompatibility is caused by the 77
negative interactions between functionally divergent genes in the parental species 78
(Dobzhansky, 1937; Muller, 1942). 79
Rice, a major food crop and model plant for genomic study of monocotyledon 80
species, also provides a model system for studying reproductive isolation. The Asian 81
cultivated rice is composed of two subspecies, indica and japonica. Their hybrids are 82
usually highly sterile, which is a barrier for utilization of the strong vigor in 83
indica-japonica hybrids. A number of loci responsible for hybrid sterility have been 84
identified, including ones that cause embryo-sac sterility, pollen sterility and spikelet 85
sterility (Ouyang et al., 2009). So far, six loci responsible for hybrid incompatibility 86
have been cloned. Among them, DPL1/DPL2 (Mizuta et al., 2010), S27/S28 87
(Yamagata et al., 2010) and Sa (Long et al., 2008) are responsible for pollen fertility, 88
while hsa1 (Kubo et al., 2016), S7 (Yu et al., 2016), and S5 (Chen et al., 2008; Yang et 89
al., 2012) control female fertility, all of them cause spikelet sterility. Three 90
evolutionary genetic models have been proposed to summarize the findings: parallel 91
divergence model, sequential divergence model and parallel-sequential divergence 92
model (Ouyang and Zhang, 2013). DPL1/DPL2 and S27/S28 in rice, HPA1/HPA2 in 93
Arabidopsis, and JYAlpha in Drosophila conform to the parallel divergence model. Sa, 94
hsa1 and many loci in other species can be explained by the sequential divergence 95
model. However, S5 in rice is the only case identified so far to follow the 96
parallel-sequential divergence model. 97
S5 is a major locus controlling indica-japonica hybrid sterility identified in many 98
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studies (Ikehashi and Araki, 1986; Liu et al., 1992; Liu et al., 1997; Wang et al., 1998; 99
Qiu et al., 2005; Song et al., 2005; Wang et al., 2005; Chen et al., 2008), and is also 100
associated with the divergence and domestication of rice (Du et al., 2011; Ouyang et 101
al., 2016). In a typical indica-japonica hybrid, the preferential abortion of female 102
gametes with japonica allele is regulated cooperatively by three adjacent genes, ORF3, 103
ORF4, and ORF5 at the S5 locus (Yang et al., 2012). Typical japonica and indica 104
varieties carry ORF3-ORF4+ORF5- and ORF3+ORF4-ORF5+, respectively, where 105
the functional allele of each gene is indicated with “+” while the non-functional allele 106
as “-”. ORF5 encodes an aspartic protease with cell wall localization. The indica 107
allele ORF5+ differs from the japonica allele ORF5- by two nucleotides, both of 108
which cause amino acid changes (Chen et al., 2008). The japonica allele ORF4+ 109
encodes an unknown transmembrane protein while the indica allele ORF4- has an 110
11-bp deletion resulting in an endoplasmic reticulum (ER)-localization. ORF3 is 111
predicted to encode a heat shock protein (HSP) resident in ER functioning to resolve 112
ER stress. The japonica allele ORF3- has a frameshift in the C terminus relative to the 113
indica ORF3+ due to a 13-bp deletion. In this system, extracellular ORF5+ 114
presumably catalyzes its substrate producing a substance that is sensed by the 115
transmembrane protein ORF4+, which induces ER stress and subsequently leading to 116
premature programed cell death (PCD). It is speculated that ORF3+, but not ORF3-, 117
can prevent/resolve the ER stress caused by ORF5+/ORF4+. Based on the results of 118
genetic and cytological analyses, Yang et al (2012) proposed a killer-protector model 119
for the roles of these three genes in this system: ORF5+, together with ORF4+, kills 120
the gametes by inducing ER stress that causes premature PCD. ORF3+ protects the 121
gametes from being killed thus restores the hybrids fertility by preventing/resolving 122
ER stress. 123
In recent years, transcriptomic analysis based on RNA-sequencing technology 124
has become a powerful tool to characterize regulatory networks and pathways to 125
enhance understanding of biological processes (Vogel et al., 2005; Digel et al., 2015; 126
Groszmann et al., 2015; Coolen et al., 2016). In this study, we performed a 127
comparative transcriptomic analysis of pistils from Balilla (a typical japonica variety), 128
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BalillaORF5+ (Balilla with a transformed ORF5+) and BalillaORF3+ORF5+ 129
(Balilla with transformed ORF3+ and ORF5+) before, during, and after the 130
megaspore meiosis. The comparisons detected thousands of genes that were 131
differentially regulated in these three genotypes at various stages. Functional 132
classification of the differentially regulated genes identified major patterns of 133
differential expression, which suggested the biological processes underlying the 134
killer-protector system of the S5 locus. These results provide insights into the 135
functional roles of ORF3+, ORF4+ and ORF5+ genes as well as the cellular and 136
biochemical nature of their interactions in regulating indica-japonica hybrid sterility. 137
138
139
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RESULTS 140
Ovule abortion process of BL5+ 141
Previous study (Yang et al., 2012) showed that the S5 reproductive barrier is 142
composed of three tightly-linked genes, ORF3, ORF4 and ORF5, in which ORF5+ 143
and ORF4+ together function as the killer of the female gametes and ORF3+ protects 144
the gametes from killing. A typical indica variety has the haplotype of ORF3+, 145
ORF4-, ORF5+ and japonica variety is of ORF3-, ORF4+, ORF5-. Balilla 146
(abbreviated BL) is a typical japonica variety. Transgenic plants BalillaORF5+ 147
(abbreviated BL5+) carrying a transformed ORF5+ under its native promoter showed 148
extremely low spikelet fertility (6.50%) compared with wild type BL (75.52%). In 149
contrast, BalillaORF3+ORF5+ (abbreviated BL3+5+), carrying both transformed 150
ORF5+ and transformed ORF3+ (also under its native promoter), had normal spikelet 151
fertility (77.35%) due to the protective role of ORF3+ (Supplemental Fig. S1A-C). 152
Paraffin section results showed that the ovule development process in both BL and 153
BL3+5+ was normal. In brief, the archesporial cell enlarged initially and developed 154
into a megaspore mother cell (Fig. 1A, K). The megaspore mother cell then 155
underwent meiotic division forming four megaspores in a line (Fig. 1B, C, L, and M). 156
The three megaspores near the micropyle degenerated, while the megaspore nearest 157
the chalaza enlarged and developed into a functional megaspore (FM) (Fig. 1D, N). 158
The FM underwent three rounds of mitotic division to produce an eight-nucleate 159
embryo-sac (Fig. 1E, O). In the aborted ovule of BL5+, the megaspore mother cell, 160
dyad and tetrad could be produced as in BL (Fig. 1F-H). However, the nucellus cells 161
showed vacuolization and even degeneration at the meiosis stage (Fig. 1G-H). After 162
meiosis, morphological abnormalities of both nucellus cells and FM were observed 163
(Fig. 1I). The abnormal FM did not enlarge and failed to develop into a mature 164
embryo-sac. The aborted embryo-sac accumulated unknown substances that were 165
deeply stained (Fig. 1J). Thus, the megaspore meiosis process in BL5+ was normal 166
but the nucellus cells around the megaspores showed serious defects at the megaspore 167
meiosis stage. The nucellus cells degradation and FM abortion caused the sterility of 168
BL5+. 169
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ORF5 and ORF4 were neighboring genes overlapping in their promoter region 170
(Yang et al., 2012). This kind of gene pairs are frequently co-expressed (Wang et al., 171
2009; Kourmpetli et al., 2013). ORF5 and ORF4 had similar expression profile and 172
relative high expression in reproductive organs (Yang et al., 2012). The in situ 173
hybridization showed that ORF5+ had localized expression in developing ovules 174
(Chen et al., 2008). Similarly, ORF4+ also displayed high and specific expression in 175
ovule tissues (Supplemental Fig. S2G-K). The specific expression of ORF5+ and 176
ORF4+ in ovules was in accordance with the specific abortion of ovules in BL5+. 177
However, the protector ORF3+ showed a wide range of expression in many tissues 178
including ovules, indicating its possible function in many processes (Supplemental 179
Fig. S2A-F). 180
Based on the ovule development process, we divided the ovule abortion process 181
into three stages (Fig. 1). The first was the megaspore mother cell (MMC) stage when 182
the ovule had megasporocyte and no obvious abnormalities were observed in BL5+. 183
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The second was the meiosis (MEI) stage when meiosis was in progress and the 184
nucellus cells of BL5+ were vacuolated. The third was after meiosis (AME) when 185
some unknown substances accumulated in embryo-sac (Fig. 1). 186
187
Processes underlying gamete abortion in BL5+ 188
We collected the pistils of wild type BL, sterile BL5+ and fertility-restored 189
BL3+5+ at MMC, MEI and AME, and performed transcriptomic analysis. A data 190
matrix of three stages from three genotypes was generated. Totally 19,269 to 20,928 191
genes were detected as expressed in these tissues at different stages representing 192
34.53% to 37.50% of total annotated genes of the Nipponbare reference genome 193
(Supplemental Table S1) (Kawahara et al., 2013). A total of 8,339, 6,278 and 530 194
genes were differentially expressed in BL5+ compared with BL at MMC, MEI and 195
AME, respectively, including 4,234, 2,430 and 410 genes that were up-regulated at 196
the three stages and 4,105, 3,848 and 120 genes that were down-regulated 197
(Supplemental Table S2; Supplemental Dataset 1). Among the differentially expressed 198
genes (DEGs), 2,067 were up-regulated and 3,325 were down-regulated at both MMC 199
and MEI (Fig. 2A). These 5,392 common DEGs accounted for 64.66% and 85.89% of 200
total DEGs at MMC and MEI, respectively, indicating that MMC and MEI involved 201
substantially overlapping cellular and molecular responses. 202
An overview of the differentially induced processes in BL5+ revealed by the 203
transcriptomes. We performed MapMan and Gene Ontology (GO) enrichment 204
analyses to classify the DEGs. MapMan is an effective tool to analyze metabolic 205
pathways and other biological processes of the transcriptome systematically (Thimm 206
et al., 2004; Ramsak et al., 2014). To get a global view of the 8,339 DEGs in BL5+ at 207
MMC, we used the “Metabolism overview” and “Cellular response” of MapMan to 208
outline the transcriptional changes of genes with putative functions in metabolism and 209
cellular response (Fig. 2B-C). 210
In “Metabolism overview”, primary metabolic processes, including cell wall (354 211
genes), amino acid (226 genes), lipids (316 genes), nucleotide (81 genes) and major 212
carbohydrate (128 genes) metabolism, accounted for 13.25% of total DEGs. The cell 213
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wall localization of ORF5+, which was previously hypothesized to initiate the 214
premature PCD (Yang et al., 2012), led us to focus our attention to the cell wall 215
metabolic genes. We found that 187 (128 up-regulated and 59 down-regulated) of the 216
354 genes were involved in cell wall degradation and modification, which may affect 217
the cell wall structures in one way or another. The secondary metabolism processes 218
including terpenes (64 genes), flavonoids (105 genes), and phenylpropanoids (120 219
genes) metabolism, which are important in plant stress response (Dixon and Paiva, 220
1995; Winkelshirley, 2002; Tholl, 2006), constituted 3.47% of all DEGs. In the term 221
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of “Cellular response”, 8.65% (721 genes) of DEGs were responsive to biotic and 222
abiotic stress; 1.94% (162 genes) of DEGs were related to redox, a phenomenon 223
usually triggered by stress; 6.67% (667 genes) of DEGs functioned in developmental 224
processes. 225
GO analysis for up-regulated genes revealed that three GO terms related to cell 226
wall metabolism, seven GO terms involved in stress or stimulus response, and eleven 227
GO terms associated with gene expression, protein synthesis and translation were 228
significantly enriched (FDR < 0.005) (Fig. 3). 229
The change in cell wall structure by cell wall modification genes may cause cell 230
wall integrity damage, which was similar to the situation of pathogen or pest attack. 231
We thus explored the “Biotic stress” in BL5+ at MMC and found that DEGs in 232
various functional categories of biotic stress were enriched including R genes, 233
pathogen-related proteins, signaling, hormone signaling, proteolysis, ROS-regulation 234
and biotic stress-responsive transcription factors (Supplemental Fig. S3A). 235
Thus, both of the MapMan and GO results indicated that a severe stress response 236
including both biotic and abiotic stresses were induced in BL5+ at MMC, which may 237
be caused by the damage in cell wall integrity, even though no abnormalities were 238
observed at this stage (Fig. 1F). 239
At MEI, 163 of the 300 DEGs in “cell wall” were related to the cell wall 240
degradation and modification including 102 up-regulated and 61 down-regulated ones 241
(Fig. 2B-C). Cell wall-related GO terms such as “glucan metabolic process”, 242
“polysaccharide metabolic process” and “polysaccharide biosynthetic process” were 243
enriched (Fig. 3). The continuous up-regulation of cell wall degrading and modifying 244
genes at MMC and MEI might be related to the observed cell wall abnormalities such 245
as vacuolization and degradation at MEI (Fig. 1G-H). In addition, 599 DEGs (9.54% 246
of total DEGs) in “biotic stress” and “abiotic stress” and genes in various functional 247
categories of biotic stress were identified (Fig. 2B, Supplemental Fig. S3B), 248
suggesting that the stress response also continued at MEI. 249
By the AME stage, DEG numbers in all the metabolic processes and cellular 250
responses were largely reduced compared to those at MMC and MEI (Fig. 2B). The 251
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ovule had aborted with the accumulation of unknown substances. In contrast, however, 252
a GO term “cellulose biosynthetic process” was significantly enriched (Fig. 3), 253
indicating misregulation of cellulose biosynthesis in BL5+. 254
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Large-scale transcriptional changes of cell wall modifying and degrading 255
genes in BL5+ at MMC and MEI. The MapMan and GO results showed that cell 256
wall metabolism especially cell wall modification and degradation were active in 257
BL5+ (Fig. 2). Cell wall modifying and degrading genes, also called cell wall 258
remodeling genes, affect cell growth by changing the extensibility and 259
physiochemical properties of the cell wall (Cosgrove, 1999; Yu et al., 2011; Gou et al., 260
2012; Hepler et al., 2013; Xiao et al., 2014). We analyzed the expression of genes 261
from seven main enriched families: expansins (EXPs), xyloglucan 262
endotransglucosylase/hydrolases (XTHs), pectin methyl esterases (PMEs), pectin 263
acetylesterases (PAEs), polygalacturonases (PGases), glycoside hydrolase family 9 264
(GH9) and arabinogalactan proteins (AGPs), which affect cell wall structure and 265
extension by enzymatic or non-enzymatic ways (Bosch et al., 2005; Coimbra et al., 266
2008; Maris et al., 2009; Gou et al., 2012; Che et al., 2016). We found that 95, 85 and 267
23 genes from these families were differentially expressed at MMC, MEI, and AME, 268
respectively, of which 73 genes were differentially expressed at both MMC and MEI, 269
including 59 up-regulated and 14 down-regulated (Fig. 4A-B). The 59 up-regulated 270
genes consisted of 13 EXPs, 7 XTHs, 5 PMEs, 4 PAEs, 8 PGases, 7 GH9s and 15 271
AGPs. OsEXP4 was elevated in BL5+ at MMC and MEI. In rice, overexpression of 272
OsEXP4 results in increased cell length, while repression of OsEXP4 reduces cell 273
length (Choi et al., 2003). Three up-regulated XTHs (Os06g48160, Os03g01800 and 274
Os10g39840) have been characterized to utilize xyloglucan, one of the cell wall 275
components, as substrate (Hara et al., 2014). OsGLU1, a GH9 member, which 276
regulates cell elongation in rice (Zhou et al., 2006; Zhang et al., 2012), was found 277
up-regulated as well. The up-regulation of genes involved in cell wall degradation and 278
cell size regulation was in accordance with cell wall abnormalities in BL5+ (Fig. 279
1G-H). Thus, we inferred that the up-regulation of the cell wall remodeling genes 280
induced by ORF5+ in BL5+ led to cell structure change, damaging the cell wall 281
integrity. 282
Cellulose deposition in BL5+. In BL5+, unknown substances were accumulated 283
in the aborted ovule at AME (Fig. 1I-J). The enrichment of GO term “cellulose 284
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biosynthetic process” suggested a misregulation of cellulose biosynthesis and that the 285
deposited substances in the aborted ovule might over-accumulate cellulose. To 286
validate this, we used Pontamine fast scarlet 4B (S4B) and calcofluor white to stain 287
cellulose (Anderson et al., 2010; Thomas et al., 2012), and observed strong 288
fluorescence signals in BL5+ at the site where the substances accumulated, indicating 289
that cellulose was indeed in large quantity (Fig. 5B, E), which was also supported by 290
the much higher cellulose content in panicles of BL5+ than in BL (Fig. 5G). In 291
contrast, normal mature embryo-sac structures were easily identified in the ovule of 292
BL and BL3+5+, where low fluorescence intensity was detected (Fig. 5A, C, D, and 293
F). 294
Cellulose is a major cell wall polysaccharide composed of unbranched β-1, 295
4-linked glucan chains. In rice, OsMYB46 and OsSWNs are secondary cell wall 296
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biosynthetic regulators. OsSWN1 acts on the upstream of OsMYB46 by binding to the 297
SNBE element in its promoter region. Overexpression of OsSWN1 or OsMYB46 in 298
Arabidopsis results in the ectopic deposition of cellulose, xylan and lignin (Zhong et 299
al., 2011). In BL5+, OsSWN1, OsSWN4, OsSWN6 and OsMYB46 were up-regulated 300
at MMC and MEI. Three cellulose synthases, OsCESA4, OsCESA7 and OsCESA9, 301
which are regarded as subunits of cellulose synthase complex (Tanaka, 2003), were 302
continuously up-regulated from MMC to AME. In addition, expression of five 303
cellulose synthase-like genes (OsCslA1, OsCslA3, OsCslA5, OsCslA9 and OsCslF6) 304
and two brittle culm genes (BC1 and BC3) were also elevated at MMC or MEI (Fig. 305
4C). Upstream transcription factors such as OsSWN1, OsSWN6 and OsMYB46 had 306
peak expression at MEI while their downstream genes such as OsCESAs and two 307
brittle culm genes had highest expression at AME (Supplemental Fig. S4), in 308
agreement with the over-accumulation of cellulose at AME (Fig. 5B, E). These results 309
showed that the cellulose biosynthetic pathway was highly activated in BL5+ 310
resulting in the accumulation of cellulose in the aborted embryo-sac. 311
Callose deposition in BL5+. Callose is a dynamic component of plant cell wall, 312
which plays important roles in megasporogenesis. The uneven distribution of the 313
callose wall around the megaspores decides the polarization of megaspores, the 314
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formation of a functional megaspore and the abortion of the other three non-functional 315
megaspores (Ünal et al., 2013). Since many genes encoding β-1,3-glucanases, which 316
have putative functions in callose metabolism, were differentially expressed in BL5+ 317
at MMC and MEI (Supplemental Fig. S5), we investigated the dynamic callose 318
distributions in ovule of BL, BL5+ and BL3+5+. During meiosis, callose 319
preferentially accumulated at the micropylar end of megaspores. Two callose bands 320
were observed in all three genotypes at the dyad stage (Fig. 6A-F), and at tetrad stage 321
at least three callose bands could be recognized also in all three genotypes although 322
slightly different in distribution patterns (Fig. 6G-L). This apparent normal callose 323
distribution in megaspores of BL5+ indicated likely normal meiosis. Unlike BL and 324
BL3+5+, however, weak callose deposition was detected in nucellus cells at the 325
tetrad stage in BL5+ (Fig. 6I). At the functional megaspore (FM) stage, callose 326
accumulated in the three degenerated gametes but not in the FM (Fig. 6M-R). The 327
survived FM subsequently developed to embryo-sac (Fig. 6S, T, W, X). However, the 328
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ovule of BL5+ displayed irregular callose deposition around the FM and in nucellus 329
cells (Fig. 6O, P), and finally, the aborted embryo-sac was stuffed with large amount 330
of callose (Fig. 6U, V). Thus, ORF5+ resulted in a misregulation of callose deposition 331
during megasporogenesis such that callose began to accumulate abnormally from MEI 332
and was deposited in large quantity at AME in the ovule of BL5+. 333
ER stress and premature PCD induced in BL5+. Under stress conditions, the 334
number of misfolded proteins will increase and may exceed the capacity of the ER 335
quality-control system, thus inducing ER stress (Howell, 2013; Wan and Jiang, 2016). 336
In BL5+, 78.95% genes in “RNA transcription”, 66.23% genes in “RNA processing”, 337
57.69% genes in “protein synthesis initiation”, 58.33% genes in “protein synthesis 338
elongation”, 82.46% genes in “ribosomal protein synthesis” and 60.91% genes in 339
“ribosome biogenesis” were up-regulated at MMC (Supplemental Fig. S6), suggesting 340
a greatly increased level of protein synthesis, which may increase the protein folding 341
burden on ER. To detect whether ER stress occurred in BL5+ at MMC, we examined 342
the expression of ER stress responsive genes in BL5+ and found that 10 such genes 343
were significantly up-regulated including OsbZIP50, which is a key ER stress 344
regulator in rice (Fig. 7A). The mRNA of OsbZIP50 was spliced when ER stress 345
occurred (Hayashi et al., 2012). We then detected the unconventional splicing of 346
OsbZIP50 mRNA in different materials and found that OsbZIP50 mRNA was spliced 347
in BL5+ at MMC, but not in BL or BL3+5+ (Fig. 7B). The spliced OsbZIP50 mRNA 348
encodes a transcription factor OsbZIP50-S, which binds to the pUPRE-II cis-element 349
in the promoter regions of OsBIP4, ORF3, OsEBS and OsPDIL2-3 (Hayashi et al., 350
2013; Takahashi et al., 2014; Wakasa et al., 2014). Accordingly, expression of these 351
four genes was elevated (Fig. 7A). Some other genes including Fes1-like, CRT2-2, 352
CRT1 and SAR1B-like, which were induced in ER stress, were also up-regulated. 353
These results showed that ER stress occurred in BL5+ at MMC relative to BL and 354
BL3+5+. In response to ER stress, genes involved in protein modification (303 355
up-regulated and 358 down-regulated) and targeting (70 up-regulated and 53 356
down-regulated) were differentially expressed presumably to help protein processing 357
and transportation. In addition, 793 DEGs (339 up-regulated and 454 down-regulated) 358
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involved in “protein degradation” were found, likely as remedy to degrade the 359
misfolded or unfolded proteins (Supplemental Fig. S6). The splicing of OsbZIP50 360
mRNA and up-regulation of ER stress responsive genes were also detected at MEI 361
and AME (Fig. 7A-B), indicating that ER stress proceeded continuously in BL5+. 362
The unresolved ER stress could induce premature programmed cell death (PCD) 363
(Cai et al., 2014). In BL5+, PCD signals were detected from the meiosis stage (Yang 364
et al., 2012), corresponding to the MEI stage in this study. At MEI, we detected 365
up-regulation of two aspartic protease-encoding genes, OsAP25 and OsAP37 (Fig. 7A, 366
C), both of which participate in the tapetal PCD process and induce PCD in yeast and 367
plants (Niu et al., 2013). Overexpression of OsAP25 or OsAP37 in plants results in 368
cell death, which is suppressed by aspartic protease-specific inhibitor pepstatin A. 369
Transformation of OsAP25 or OsAP37 into a PCD-deficient yeast strain restores the 370
PCD ability of the strain. OsAP25 showed the highest expression at MEI (Fig. 7C), 371
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consistent with the observation of PCD signals at this stage. In tapetal PCD process, 372
transcription factor EAT1 directly regulates the expression of OsAP25 and OsAP37 373
(Niu et al., 2013). However, EAT1 was down-regulated at MEI, indicating that some 374
other regulators might have replaced EAT1 to activate OsAP25 and OsAP37. TDR and 375
TIP2, which work upstream of OsAP25 and OsAP37 in pollen development (Niu et al., 376
2013), were up-regulated in BL5+ at MMC and MEI (Fig. 7A, D). The coordinated 377
up-regulation of TDR, TIP2, OsAP25 and OsAP37 suggested that the ovule and 378
tapetum may share some common genes/pathways in PCD. Taken together, an 379
OsbZIP50-dependent ER stress pathway and a premature PCD pathway involving the 380
tapetal PCD genes were activated in BL5+. 381
These results suggested that the ORF5+-induced up-regulation of cell wall 382
degradation and modification genes in BL5+ caused cell wall damage, which induced 383
both biotic and abiotic stresses. Lasting stress responses induced ER stress and 384
unresolved ER stress led to premature PCD eventually resulting in the ovule abortion. 385
Processes detected in the BL3+5+ /BL comparison. 386
BL3+5+ carrying both the killer ORF5+ and the protector ORF3+ showed no 387
phenotypic difference from the wild type BL with respect to fertility (Supplemental 388
Fig. S1). However, 3,986 DEGs were still identified between BL3+5+ and BL at 389
MMC (Supplemental Table S2; Supplemental Dataset 2), of which 3,843 DEGs were 390
shared with the BL5+/BL comparison at this stage, indicating that ORF5+ induced 391
similar processes in BL3+5+ (Fig. 8A). As expected, the process of “cell wall” was 392
enriched with 217 DEGs (Fig. 8B), of which 64 (55 up-regulated and 9 393
down-regulated) were cell wall modifying and degrading genes from the seven main 394
families (Fig. 4A-B). GO terms “glucan metabolic process” and “polysaccharide 395
metabolic process” were significantly enriched in up-regulated genes (Fig. 8C). Terms 396
“biotic stress” and “abiotic stress” were also enriched involving 339 DEGs. However, 397
the number of DEGs in each of the enriched functional categories was smaller than 398
that in the BL5+/BL comparison (Fig. 4A-B). Moreover, the splicing of OsbZIP50 399
mRNA and the up-regulation of its downstream genes were not detected (Fig. 7A-B). 400
These results suggested that ORF5+ induced similar processes in BL3+5+ at MMC 401
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especially with respect to cell wall integrity damage, but to a much lesser extent, 402
which did not trigger ER stress. 403
At MEI, the difference between BL3+5+ and BL was greatly narrowed that only 404
749 DEGs were identified, compared with 6,278 genes in the BL5+/BL comparison 405
(Supplemental Table S2; Supplemental Dataset 2). Thus numbers of DEGs especially 406
those in cell wall, biotic stress and abiotic stress were greatly reduced (Fig. 8B). Only 407
7 DEGs from the seven cell wall families were found (Fig. 4A-B), suggesting that cell 408
wall integrity damage was suppressed. The cell wall compensation pathways such as 409
cellulose biosynthesis and callose deposition were not activated (Fig 4C; Fig. 6K, Q), 410
and ER stress and PCD did not occur (Fig. 7). As a result, the cell wall maintained 411
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integrity and the ovule meiosis was undisturbed (Fig. 1L-M). 412
At AME, the number of DEGs was further reduced to 370 in BL3+5+ compared 413
with BL (Supplemental Table S2; Supplemental Dataset 2). These results indicated 414
that ORF5+ induced similar but weaker damage processes in BL3+5+ as in BL5+ at 415
MMC, but these processes were soon suppressed by ORF3+. Consequently, the 416
embryo-sac developed normally producing fertile female gametes (Fig. 1O). 417
418
419
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DISCUSSION 420
The killer-protector system of S5 is composed of three proteins, cell wall protein 421
ORF5+, transmembrane protein ORF4+, and ER resident protein ORF3+. Based on 422
the comparative transcriptome results of BL5+ and BL3+5+, we proposed a model to 423
summarize the processes underlying the S5 system (Fig. 9). 424
In BL5+, many cell wall modifying and degrading genes in the families EXPs, 425
XTHs, PMEs, PAEs, PGases, GH9, and AGPs were up-regulated at MMC and MEI, 426
and many genes of these families play important roles in cell elongation or cell 427
expansion (Zhou et al., 2006; Chen and Ye, 2007; Yang et al., 2007; Coimbra et al., 428
2008; Maris et al., 2009; Zhang et al., 2010; Yu et al., 2011; Gou et al., 2012; Zhang et 429
al., 2012; Ma et al., 2013; Miedes et al., 2013; Hara et al., 2014; Xiao et al., 2014; 430
Che et al., 2016). It has been suggested that post-synthetic modifications of the cell 431
wall by cell wall modifying genes may affect cell wall integrity (CWI) (Pogorelko et 432
al., 2013). Thus, large-scale up-regulation of cell wall remodeling genes induced by 433
ORF5+ may lead to changes of cell wall structures, which affect CWI thus inducing 434
stress response. 435
ORF5+ encodes an extracellular aspartic protease. In yeast, extracellular aspartic 436
proteases yapsins are required for the CWI maintenance (Krysan et al., 2005; Guan et 437
al., 2012). Two plant aspartic proteases A36 and A39 participate in cell wall 438
remodeling in pollen tube (Gao et al., 2017). The study in Candida albicans shows 439
that secreted aspartic proteinases use a set of cell wall proteins as substrates (Schild et 440
al., 2011). We speculated that one or more substrates of ORF5+ existed in the rice cell 441
wall, which induced cell wall damage after activated by ORF5+. In yeast, CWI 442
damage triggers unfolded protein response (UPR), a homeostatic response to alleviate 443
ER stress, which in turn affects the CWI (Krysan, 2009; Scrimale et al., 2009). In 444
plants, pathogen invasion or pest attack can induce CWI damage and subsequent 445
stress response (Bellincampi et al., 2014; Pogorelko et al., 2013). CWI damage 446
eventually leads to PCD (Paparella et al., 2015). In BL5+, the functional categories 447
responsive to pathogen invasion or pest attack were enriched, and ER stress was 448
accordingly induced presumably by CWI impairment, which in turn exacerbated the 449
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cell wall damage and its subsequent stress responses, thus finally leading to PCD. To 450
alleviate the adverse effects caused by CWI damage, cells usually trigger a cell wall 451
compensatory pathway to strengthen the cell wall (Hamann, 2012). For example, 452
disruption by aspartic protease Yps7p in Pichia pastoris reduces chitin content in the 453
lateral cell wall, but the cell thickens the inner cell wall for compensation (Guan et al., 454
2012). Similarly in BL5+, key genes in secondary cell wall biosynthesis were 455
significantly up-regulated resulting in the cellulose accumulation to repair cell wall. 456
Callose, a polysaccharide usually accumulated under stress conditions to provide 457
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physical barrier to the adverse environments (Pirselova and Matusikova, 2012), was 458
deposited in large quantity. Despite other roles of callose in ovule development, the 459
large amount deposition of callose in BL5+ was most likely to strengthen cell wall. 460
ORF4+ acted as a partner of ORF5+ with a transmembrane localization. We 461
speculated that ORF4+ functioned as a sensor of CWI damage. In plants, a 462
well-studied cell wall sensor is AtWAK1, a wall-associated kinase, which resides in 463
the plasma membrane and responds to oligogalacturonides derived from the 464
hydrolysis of cell wall component (Brutus et al., 2010). Xa4, a wall-associated kinase 465
in rice, contributes to disease resistance by strengthening the cell wall via promoting 466
cellulose synthesis and suppressing cell wall loosening (Hu et al., 2017). Unlike 467
wall-associated kinase, no extracellular binding domain or inner kinase domain was 468
found in ORF4+, thus there may exist a co-receptor that interacts with ORF4+ in the 469
plasma membrane and/or a downstream signal protein that interacts with plasma 470
membrane receptor to transmit signals. 471
Based on these analyses, it was deduced that ORF5+ damaged CWI by cleaving 472
its substrates leading to up-regulation of the cell wall modifying and degrading genes. 473
ORF4+ sensed the CWI impairment and induced biotic and abiotic stress responses, 474
consequently induced ER stress due to the accumulation of drastically increased 475
misfolded proteins in ER. The occurrence of ER stress in turn deteriorated the CWI 476
damage and led to a more serious stress response. Finally, the unresolved cell wall 477
damage, stress response and ER stress triggered PCD. Meanwhile, the cellulose and 478
callose accumulated in ovule as the cells’ unsuccessful remedy to repair the damaged 479
cell wall. 480
BL3+5+ showed no difference with BL with respect to ovule development and 481
fertility, but a number of cell wall modifying and degrading genes were still 482
up-regulated in BL3+5+ at MMC, which may induce CWI damage. However, 483
transcriptional change of these genes was suppressed at MEI and AME. Thus, the 484
subsequent negative effects caused by CWI damage including ER stress, PCD, 485
cellulose and callose deposition were prevented in BL3+5+. ORF3+ encodes a heat 486
shock protein 70 chaperone, which is a downstream target of OsbZIP50s and induced 487
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25
by ER stress. ORF3+ is believed to help protein folding in ER to resolve ER stress 488
like OsBIP1-OsBIP5 in the HSP70 gene family (Yang et al., 2012; Hayashi et al., 489
2013; Wakasa et al., 2014). BIP protein alleviates Cd2+ stress-induced PCD (Xu et al., 490
2013). The tobacco plants with overexpression of BIP show delayed leaf senescence 491
(Carvalho et al., 2014). In BL3+5+, ORF3+ suppressed the ORF5+-induced ER 492
stress and PCD. It was speculated that one role of ORF3+ was alleviating ER stress 493
thus not to induce premature PCD. The chaperone activity of ORF3+ may also help 494
the folding of the signal proteins downstream of ORF5+ and ORF4+ so that the stress 495
response may be suppressed at MEI. 496
In conclusion, we revealed the processes underlying a reproductive barrier 497
composed of ORF3, ORF4, and ORF5 at the transcriptomic level and proposed a 498
mechanistic model based on the results. Nevertheless, further studies are needed to 499
support and enrich the model. 500
501
MATERIALS AND METHODS 502
Sample preparation and library construction 503
Balilla was a typical japonica rice variety. The transgenic plant BalillaORF5+ 504
was generated by introducing ORF5+ from an indica variety Nanjing 11 into Balilla 505
under its native promoter (Chen et al., 2008). The plant BalillaORF3+ORF5+ was 506
isolated from the hybrid offspring between BalillaORF5+ and BalillaORF3+, which 507
contained a transformed ORF3+ from Nanjing 11 under its native promoter (Yang et 508
al., 2012). All the rice plants were grown in the fields of the experimental station of 509
Huazhong Agricultural University, Wuhan, China. We determined the relationship 510
between the floret length and stage of ovule development by the paraffin section 511
observation of florets from the variety Balilla. The pistils from florets of Balilla, 512
BalillaORF5+ and BalillaORF3+ORF5+ in length ranges of 3.8-4.2 mm, 5.5-6.0 mm, 513
and 7.0-7.5 mm were collected as MMC, MEI and AME stage samples with two 514
biological replicates. RNA was extracted using TRIzol reagent (Invitrogen) according 515
to the manufacture’s manual. RNA yields and quality were measured using Nanodrop 516
2000 (Thermo Scientific), and further quantified using Qubit2.0 Fluorometer 517
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26
(Invitrogen). RNA integrity was evaluated using Experion RNA Analysis Kits 518
(Bio-rad) and Experion Automated Electrophoresis Station (Bio-rad). Each library 519
was constructed using 3 μg total RNA using the TruSeq RNA Library Preparation Kit 520
v2 (Illumina). The libraries were sequenced using Illumina HiSeq 2000 sequencing 521
platform at National Key Laboratory for Crop Genetic Improvement in Huazhong 522
Agricultural University, Wuhan, China. 523
Data processing and DEGs identification 524
The data of the two replicates of all samples were collected, and analyzed 525
following the workflow described previously (Trapnell et al., 2012). In brief, the raw 526
sequencing reads were mapped to the rice reference genome (MSU7.0, 527
http://rice.plantbiology.msu.edu/index.shtml) using TopHat, then the transcripts were 528
assembled and quantified using Cufflinks, and finally, differentially expressed genes 529
were analyzed using Cuffdiff. The raw data files can be found in the NCBI database 530
under the accession numbers GSE95200. Genes with ≥ 2-fold expression 531
up-regulation or down-regulation and adjusted p-value ≤ 0.05 (counted using Cuffdiff) 532
were regarded as up-regulated or down-regulated genes. 533
GO and MapMan analysis 534
Gene ontology (GO) enrichment was performed in AgriGO, a web-based tool for 535
gene ontology analysis (Du et al., 2010) (http://bioinfo.cau.edu.cn/agriGO/index.php). 536
The species “Oryza sativa” were chosen. GO terms in biological process groups with 537
FDR < 0.005 were identified and analyzed further. The MapMan software version 538
3.5.1.R2 and pathways were downloaded from MapMen Site of analysis 539
(http://mapman.gabipd.org/web/guest/mapmanstore). The mapping information of 540
rice genes (osa_MSU_v7_2015-09-08_mapping.txt.tar.gz) that can be imported to the 541
MapMan software was downloaded from GoMapMan 542
(http://www.gomapman.org/export/current/mapman) (Ramsak et al., 2014). 543
In situ hybridization 544
The methods for preparation of paraffin embedded sections followed the 545
previously described protocol (Weng et al., 2014). Primer pairs (listed in 546
Supplemental Table S4) ORF3insituF and ORF3insituR, ORF4insituF and 547
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ORF4insituR were used to prepare probes for ORF3 and ORF4, respectively. Products 548
amplified from ORF3+ or ORF4+ cDNA were ligated to pGEM-T vector (Promega). 549
The sense probe was transcribed with T7 RNA polymerase, and the antisense probe 550
was transcribed with SP6 RNA polymerase. The probes were labeled with 551
digoxigenin (Roche). Hybridization and detection were performed as described 552
previously (De Block and Debrouwer, 1993). 553
PCR and RT-qPCR 554
Total RNA of pistils were isolated with TRIzol reagent (Invitrogen) and treated 555
with RNase-free DNaseI (Invitrogen) for 20 min at room temperature to digest the 556
contaminating DNA. The first-strand cDNA was synthesized using SSIII reverse 557
transcriptase (Invitrogen) with Oligo(dT)15 Primer (Promega) following the 558
manufacturers’ instruction. For amplification of OsbZIP50s, specific primers around 559
the splice site were used. PCR conditions and PCR products detection were performed 560
as described before (Yang et al., 2012). For quantitative real-time PCR, we carried out 561
the reaction in a total volume of 10 μl containing 5 μl FastStart Universal SYBR 562
Green Master (Rox) (Roche), 1 μl cDNA sample, 3 μM each primer on ABI ViiA™7 563
system. The primers for PCR and RT-qPCR are listed in Supplemental Table S4. The 564
gene profilin (Os06g05880) was used as the internal control in RT-qPCR (Yang et al., 565
2012). 566
Histological staining 567
The fresh florets were fixed with FAA solution (50% ethanol, 5% acetic acid, 3.7% 568
formaldehyde) at 4°C overnight, dehydrated with ethanol in a series of concentrations 569
(50%, 70%, 85%, 95%, 100%), infiltrated with xylene and finally embedded in 570
paraffin. The materials were cut into 10 μm-thick sections and then washed in a 571
xylene/ethanol series (0–50% ethanol). For callose staining, the rehydrated sections 572
were stained with 0.1% aniline blue solution for 60 min. For cellulose staining, the 573
rehydrated sections were stained with 0.01% Pontamine Fast Scarlet 4B solution for 574
20 min or 0.1% calcofluor white (Sigma) for 5 min. The stained sections were 575
scanned under fluorescence microscopy. The florets for observation of ovule 576
development process were stained with ehrlich haematoxylin. After fixation, the 577
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28
materials were rehydrated, infiltrated with ehrlich haematoxylin for four days, then 578
dehydrated with ethanol, infiltrated with xylene and embedded in paraffin. The 10 579
μm-thick sections were dewaxed with xylene and sealed with Canada balsam (Sigma). 580
The slides were scanned under light microscopy. 581
Cellulose content determination 582
The cellulose content was determined as described previously (Guo et al., 2014). 583
In brief, the materials were suspended with potassium phosphate buffer (pH 7.0), 584
chloroform-methanol (1:1, v/v), dimethylsulphoxide (DMSO)-water (9:1, v/v), 0.5% 585
(w/v) ammonium oxalate, 4 M KOH containing 1.0 mg/mL sodium borohydride in 586
turn. The remaining pellet was collected and extracted further with H2SO4 (67%, v/v) 587
and the supernatants were collected for the determination of cellulose. 588
589
SUPPLEMENTAL DATA 590
Supplemental Figure S1. The fertility of BL, BL5+, and BL3+5+. 591
Supplemental Figure S2. RNA in situ hybridization of ORF3+ and ORF4+. 592
Supplemental Figure S3. DEGs in “Biotic stress” in BL5+ compared with BL at 593
MMC (A) and MEI (B). 594
Supplemental Figure S4. qPCR verification for expression of the cellulose 595
biosynthetic genes in pistils of BL, BL5+, and BL3+5+ at MMC, MEI, and AME, 596
respectively. 597
Supplemental Figure S5. Expression change of genes involved in callose 598
metabolism. 599
Supplemental Figure S6. The number of DEGs involved in RNA transcription, 600
protein synthesis and processing. 601
Supplemental Table S1. The number of expressed genes in BL, BL5+, and BL3+5+ 602
at different stages. 603
Supplemental Table S2. The number of differentially expressed genes identified in 604
different comparisons. 605
Supplemental Table S3. Full list of significantly enriched GO terms (FDR < 0.005) 606
for DEGs in BL5+ vs. BL. 607
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29
Supplemental Table S4. Full list of significantly enriched GO terms (FDR < 0.005) 608
for DEGs in BL3+5+ vs. BL. 609
Supplemental Table S5. The list of cell wall modifying or degrading genes and their 610
expression changes based on log2 fold. 611
Supplemental Table S6. The primers used in this study. 612
Supplemental Dataset 1. The differentially expressed genes in BL5+ compared with 613
BL. 614
Supplemental Dataset 2. The differentially expressed genes in BL3+5+ compared 615
with BL. 616
617
ACKNOWLEDGMENTS 618
We thank Yihua Zhou from Institute of Genetics and Developmental Biology for 619
kindly providing the Pontamine Fast Scarlet 4B solution. 620
621
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30
622
FIGURE LEGENDS 623
Figure 1. The ovule development process of BL, BL5+, and BL3+5+. 624
(A-E) Ovule development of BL (gametes fertile). (F-J) Ovule development of BL5+ 625
(gametes sterile). (K-O) Ovule development of BL3+5+ (gametes fertile). (A, F, K) 626
The megaspore mother cell stage. (B, G, L) The dyad stage. (C, H, M) The tetrad 627
stage. (D, I, N) The functional megaspore stage. (E, J, O) The mature embryo sac. 628
MMC, megaspore mother cell; Dy, dyad; Te, tetrad; FM, functional megaspore; DM, 629
degenerated megaspore; AFM, aborted functional megaspore; ANC, abnormal 630
nucellus cells; ES, embryo sac; AES, aborted embryo sac. Bars = 30 μm. The grey 631
bars at the top indicate the corresponding stage in transcriptome data. MMC indicated 632
the megaspore mother cell stage. MEI indicates the meiosis stage. AME indicates the 633
stage after meiosis. 634
635
Figure 2. Analysis of genes differentially expressed in BL5+ compared to BL. 636
(A) Numbers of the up- and down-regulated DEGs at MMC and MEI. (B) Numbers of 637
DEGs in different MapMan functional categories at MMC, MEI and AME, 638
respectively. (C) The heat map of “Metabolism overview” and “Cellular response” in 639
BL5+ at MMC based on the MapMan results. Each small square represents a gene 640
differentially expressed in BL5+ vs. BL. The color of the square represents the level 641
of up-regulation (red) or down-regulation (green) based on the log2 fold change of the 642
DEGs. 643
644
Figure 3. Partial significantly (FDR < 0.005) enriched GO terms for up-regulated 645
genes in BL5+ compared to BL. 646
The color in each cell represents the significance of enrichment based on the FDR 647
value. The cells without color indicate not significantly enriched. The full list of GO 648
terms is given in Supplemental Table S3. 649
650
Figure 4. Differential regulation of cell wall genes in BL5+ and BL3+5+ compared 651
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31
with BL. 652
(A) The number of differentially expressed genes involved in cell wall modification 653
and degradation in BL5+ and BL3+5+. The X-axis indicates the gene numbers. The 654
Y-axis indicates the gene families at different stages. (B) Heat map of expression 655
change of genes involved in cell wall modification and degradation. The color in each 656
cell represents the level of expression change based on the log2 fold. The cell without 657
color indicates the gene was not differentially expressed. Full list of genes and their 658
expressions is given in Supplemental Table S5. (C) Expression change of cellulose 659
biosynthetic genes. 660
661
Figure 5. Cellulose deposition in the mature ovules of BL, BL5+, and BL3+5+. 662
(A-F) Cellulose deposition in BL (A, D), BL5+ (B, E) and BL3+5+ (C, F). (A-C) 663
S4B staining. (D-F) Calcofluor white staining. The strong red or blue fluorescence 664
indicates where the cellulose accumulated. Bars = 50 μm. (G) Cellulose content in the 665
panicles of BL and BL5+. The data are means ± SEM (n = 3). *** significant 666
difference at P < 0.001. 667
668
Figure 6. Callose deposition (green fluorescence) in the developing ovule of BL, 669
BL5+, and BL3+5+ by aniline blue staining. 670
(A-F) The dyad stage. The red arrowheads indicate the callose bands around the dyad. 671
(G-L) The tetrad stage. The red arrowheads indicate the callose bands around the 672
tetrad. (M-R) The functional megaspore stage. The red arrowheads indicate the 673
degenerated megaspores. (S-X) The mature embryo-sac. (I, O, U) The grey 674
arrowheads indicate abnormally deposited callose. Bars = 25 μm. 675
676
Figure 7. Differential regulation of ER stress and PCD genes. 677
(A) Log2 fold change of the expression level of ER stress and PCD genes in BL5+ 678
against BL. The color in each cell represents the levels of expression change based on 679
the log2 fold. The cell without color indicates the gene was not differentially 680
expressed. (B) Splicing of OsbZIP50 mRNA in BL, BL5+ and BL3+5+ at each stage. 681
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32
(C) qPCR verification for expression of tapetal PCD-related genes in BL and BL5+. 682
The X-axis represents different stages. The Y-axis represents the expression level 683
relative to profilin (Os06g05880). The data are means ± SEM (n = 3). 684
685
Figure 8. Analysis of genes differentially expressed in BL3+5+ compared to BL. 686
(A) Number of DEGs detected in the BL3+5+/BL and BL5+/BL comparisons at 687
different stages. The overlapping areas indicate the genes differentially expressed in 688
both BL3+5+ and BL5+. (B) The number of DEGs in different functional categories 689
of “Metabolism Overview” and “Cellular response” by MapMan at MMC, MEI and 690
AME, respectively. (C) Partial significantly (FDR < 0.005) enriched GO terms for 691
up-regulated genes in BL3+5+ compared with BL. The color in each cell represents 692
the significance of enrichment based on the FDR value. The cells without color 693
indicate not significantly enriched. The full list of GO terms is given in Supplemental 694
Table S4. 695
696
Fig. 9. A model for S5 locus-mediated ovule abortion. 697
698
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699
700
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