Download Reproductive barriers in indica-japonica rice

Plant Physiology Preview. Published on May 8, 2017, as DOI:10.1104/pp.17.00093
1
Running Head:
2
Transcriptomic basis of a rice reproductive barrier
3
4
Corresponding Author:
5
Qifa Zhang
6
National Key Laboratory of Crop Genetic Improvement and National Center of Plant
7
Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China
8
Phone: 86-27-87282429
9
Fax: 86-27-87287092
10
E-mail: [email protected]
11
12
Research Area:
13
Genes, Development and Evolution
14
1
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
Copyright 2017 by the American Society of Plant Biologists
15
Title of article:
16
Reproductive barriers in indica-japonica rice hybrids are uncovered by transcriptome
17
analysis
18
All authors’ full names:
19
Yanfen Zhu, Yiming Yu, Ke Cheng, Yidan Ouyang, Jia Wang, Liang Gong, Qinghua
20
Zhang, Xianghua Li, Jinghua Xiao and Qifa Zhang*
21
*For correspondence (Fax: 86-27-87287092; Phone: 86-27-87282429; e-mail:
22
[email protected])
23
24
Author contributions:
25
Y.Z. and Qifa Zhang designed the research; Y.Z., Y.Y., K.C., Y.O., J.W., L.G. and
26
Qinghua Zhang performed the research; X.L. and J.X. contributed reagents; Y.Z. and
27
Qifa Zhang analyzed data and wrote the paper.
28
The authors declare no conflict of interests.
29
30
Affiliations:
31
National Key Laboratory of Crop Genetic Improvement and National Center of Plant
32
Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China
33
34
One Sentence Summary:
35
A reproductive barrier causes female gamete abortion of indica-japonica rice hybrids
36
by damaging cell wall integrity inducing massive biotic and abiotic stresses resulting
37
in ER stress leading to premature PCD.
38
39
Footnotes:
40
Financial source: This work was supported by grants from the National Key Research
41
and Development Program (2016YFD0100903) and National Natural Science
42
Foundation (31130032) of China.
43
2
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
44
ABSTRACT
45
46
In rice (Oryza sativa L.), hybrids between indica and japonica subspecies are usually
47
highly sterile, which provides a model system for studying postzygotic reproductive
48
isolation. A killer-protector system S5 composed of three adjacent genes, ORF3,
49
ORF4 and ORF5, regulates female gamete fertility of indica-japonica hybrids. To
50
characterize the processes underlying this system, we performed transcriptomic
51
analyses of pistils from rice variety Balilla (BL), Balilla with transformed ORF5+
52
(BL5+) producing sterile female gametes, and Balilla with transformed ORF3+ and
53
ORF5+ (BL3+5+) producing fertile gametes. RNA sequencing of tissues collected
54
before (MMC), during (MEI), and after (AME) meiosis of the megaspore mother cell
55
detected 19,269 to 20,928 genes as expressed. Comparison between BL5+ and BL
56
showed that ORF5+ induced differential expression of 8,339, 6,278 and 530 genes at
57
MMC, MEI, and AME. At MMC, large-scale differential expression of cell wall
58
modifying genes and biotic and abiotic response genes indicated that cell wall
59
integrity damage induced severe biotic and abiotic stresses. The processes continued
60
to MEI and induced ER stress as indicated by differential expression of ER stress
61
responsive genes, leading to PCD at MEI and AME, resulting in abortive female
62
gametes. In the BL3+5+/BL comparison, 3,986, 749 and 370 genes were
63
differentially expressed at MMC, MEI and AME. Large numbers of cell wall
64
modification and biotic and abiotic response genes were also induced at MMC but
65
largely suppressed at MEI without inducing ER stress and PCD, producing fertile
66
gametes. These results have general implications for the understanding of biological
67
processes underlying reproductive barriers.
68
3
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
69
INTRODUCTION
70
Reproductive isolation, a phenomenon widely existing in natural organisms,
71
promotes speciation and maintains the integrity of species over time (Coyne and Orr,
72
2004). It is divided into two general categories depending on whether it occurs before
73
or after fertilization: prezygotic reproductive isolation and postzygotic reproductive
74
isolation (Seehausen et al., 2014). The former prevents the formation of hybrid
75
zygotes, while the latter results in hybrid incompatibility including hybrid necrosis,
76
weakness, sterility, and lethality in F1 or later generations (Ouyang and Zhang, 2013).
77
The Dobzhansky-Muller model suggests that hybrid incompatibility is caused by the
78
negative interactions between functionally divergent genes in the parental species
79
(Dobzhansky, 1937; Muller, 1942).
80
Rice, a major food crop and model plant for genomic study of monocotyledon
81
species, also provides a model system for studying reproductive isolation. The Asian
82
cultivated rice is composed of two subspecies, indica and japonica. Their hybrids are
83
usually highly sterile, which is a barrier for utilization of the strong vigor in
84
indica-japonica hybrids. A number of loci responsible for hybrid sterility have been
85
identified, including ones that cause embryo-sac sterility, pollen sterility and spikelet
86
sterility (Ouyang et al., 2009). So far, six loci responsible for hybrid incompatibility
87
have been cloned. Among them, DPL1/DPL2 (Mizuta et al., 2010), S27/S28
88
(Yamagata et al., 2010) and Sa (Long et al., 2008) are responsible for pollen fertility,
89
while hsa1 (Kubo et al., 2016), S7 (Yu et al., 2016), and S5 (Chen et al., 2008; Yang et
90
al., 2012) control female fertility, all of them cause spikelet sterility. Three
91
evolutionary genetic models have been proposed to summarize the findings: parallel
92
divergence model, sequential divergence model and parallel-sequential divergence
93
model (Ouyang and Zhang, 2013). DPL1/DPL2 and S27/S28 in rice, HPA1/HPA2 in
94
Arabidopsis, and JYAlpha in Drosophila conform to the parallel divergence model. Sa,
95
hsa1 and many loci in other species can be explained by the sequential divergence
96
model. However, S5 in rice is the only case identified so far to follow the
97
parallel-sequential divergence model.
98
S5 is a major locus controlling indica-japonica hybrid sterility identified in many
4
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
99
studies (Ikehashi and Araki, 1986; Liu et al., 1992; Liu et al., 1997; Wang et al., 1998;
100
Qiu et al., 2005; Song et al., 2005; Wang et al., 2005; Chen et al., 2008), and is also
101
associated with the divergence and domestication of rice (Du et al., 2011; Ouyang et
102
al., 2016). In a typical indica-japonica hybrid, the preferential abortion of female
103
gametes with japonica allele is regulated cooperatively by three adjacent genes, ORF3,
104
ORF4, and ORF5 at the S5 locus (Yang et al., 2012). Typical japonica and indica
105
varieties carry ORF3-ORF4+ORF5- and ORF3+ORF4-ORF5+, respectively, where
106
the functional allele of each gene is indicated with “+” while the non-functional allele
107
as “-”. ORF5 encodes an aspartic protease with cell wall localization. The indica
108
allele ORF5+ differs from the japonica allele ORF5- by two nucleotides, both of
109
which cause amino acid changes (Chen et al., 2008). The japonica allele ORF4+
110
encodes an unknown transmembrane protein while the indica allele ORF4- has an
111
11-bp deletion resulting in an endoplasmic reticulum (ER)-localization. ORF3 is
112
predicted to encode a heat shock protein (HSP) resident in ER functioning to resolve
113
ER stress. The japonica allele ORF3- has a frameshift in the C terminus relative to the
114
indica ORF3+ due to a 13-bp deletion. In this system, extracellular ORF5+
115
presumably catalyzes its substrate producing a substance that is sensed by the
116
transmembrane protein ORF4+, which induces ER stress and subsequently leading to
117
premature programed cell death (PCD). It is speculated that ORF3+, but not ORF3-,
118
can prevent/resolve the ER stress caused by ORF5+/ORF4+. Based on the results of
119
genetic and cytological analyses, Yang et al (2012) proposed a killer-protector model
120
for the roles of these three genes in this system: ORF5+, together with ORF4+, kills
121
the gametes by inducing ER stress that causes premature PCD. ORF3+ protects the
122
gametes from being killed thus restores the hybrids fertility by preventing/resolving
123
ER stress.
124
In recent years, transcriptomic analysis based on RNA-sequencing technology
125
has become a powerful tool to characterize regulatory networks and pathways to
126
enhance understanding of biological processes (Vogel et al., 2005; Digel et al., 2015;
127
Groszmann et al., 2015; Coolen et al., 2016). In this study, we performed a
128
comparative transcriptomic analysis of pistils from Balilla (a typical japonica variety),
5
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
129
BalillaORF5+ (Balilla with a transformed ORF5+) and BalillaORF3+ORF5+
130
(Balilla with transformed ORF3+ and ORF5+) before, during, and after the
131
megaspore meiosis. The comparisons detected thousands of genes that were
132
differentially regulated in these three genotypes at various stages. Functional
133
classification of the differentially regulated genes identified major patterns of
134
differential expression, which suggested the biological processes underlying the
135
killer-protector system of the S5 locus. These results provide insights into the
136
functional roles of ORF3+, ORF4+ and ORF5+ genes as well as the cellular and
137
biochemical nature of their interactions in regulating indica-japonica hybrid sterility.
138
139
6
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
140
RESULTS
141
Ovule abortion process of BL5+
142
Previous study (Yang et al., 2012) showed that the S5 reproductive barrier is
143
composed of three tightly-linked genes, ORF3, ORF4 and ORF5, in which ORF5+
144
and ORF4+ together function as the killer of the female gametes and ORF3+ protects
145
the gametes from killing. A typical indica variety has the haplotype of ORF3+,
146
ORF4-, ORF5+ and japonica variety is of ORF3-, ORF4+, ORF5-. Balilla
147
(abbreviated BL) is a typical japonica variety. Transgenic plants BalillaORF5+
148
(abbreviated BL5+) carrying a transformed ORF5+ under its native promoter showed
149
extremely low spikelet fertility (6.50%) compared with wild type BL (75.52%). In
150
contrast, BalillaORF3+ORF5+ (abbreviated BL3+5+), carrying both transformed
151
ORF5+ and transformed ORF3+ (also under its native promoter), had normal spikelet
152
fertility (77.35%) due to the protective role of ORF3+ (Supplemental Fig. S1A-C).
153
Paraffin section results showed that the ovule development process in both BL and
154
BL3+5+ was normal. In brief, the archesporial cell enlarged initially and developed
155
into a megaspore mother cell (Fig. 1A, K). The megaspore mother cell then
156
underwent meiotic division forming four megaspores in a line (Fig. 1B, C, L, and M).
157
The three megaspores near the micropyle degenerated, while the megaspore nearest
158
the chalaza enlarged and developed into a functional megaspore (FM) (Fig. 1D, N).
159
The FM underwent three rounds of mitotic division to produce an eight-nucleate
160
embryo-sac (Fig. 1E, O). In the aborted ovule of BL5+, the megaspore mother cell,
161
dyad and tetrad could be produced as in BL (Fig. 1F-H). However, the nucellus cells
162
showed vacuolization and even degeneration at the meiosis stage (Fig. 1G-H). After
163
meiosis, morphological abnormalities of both nucellus cells and FM were observed
164
(Fig. 1I). The abnormal FM did not enlarge and failed to develop into a mature
165
embryo-sac. The aborted embryo-sac accumulated unknown substances that were
166
deeply stained (Fig. 1J). Thus, the megaspore meiosis process in BL5+ was normal
167
but the nucellus cells around the megaspores showed serious defects at the megaspore
168
meiosis stage. The nucellus cells degradation and FM abortion caused the sterility of
169
BL5+.
7
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
170
ORF5 and ORF4 were neighboring genes overlapping in their promoter region
171
(Yang et al., 2012). This kind of gene pairs are frequently co-expressed (Wang et al.,
172
2009; Kourmpetli et al., 2013). ORF5 and ORF4 had similar expression profile and
173
relative high expression in reproductive organs (Yang et al., 2012). The in situ
174
hybridization showed that ORF5+ had localized expression in developing ovules
175
(Chen et al., 2008). Similarly, ORF4+ also displayed high and specific expression in
176
ovule tissues (Supplemental Fig. S2G-K). The specific expression of ORF5+ and
177
ORF4+ in ovules was in accordance with the specific abortion of ovules in BL5+.
178
However, the protector ORF3+ showed a wide range of expression in many tissues
179
including ovules, indicating its possible function in many processes (Supplemental
180
Fig. S2A-F).
181
Based on the ovule development process, we divided the ovule abortion process
182
into three stages (Fig. 1). The first was the megaspore mother cell (MMC) stage when
183
the ovule had megasporocyte and no obvious abnormalities were observed in BL5+.
8
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
184
The second was the meiosis (MEI) stage when meiosis was in progress and the
185
nucellus cells of BL5+ were vacuolated. The third was after meiosis (AME) when
186
some unknown substances accumulated in embryo-sac (Fig. 1).
187
188
Processes underlying gamete abortion in BL5+
189
We collected the pistils of wild type BL, sterile BL5+ and fertility-restored
190
BL3+5+ at MMC, MEI and AME, and performed transcriptomic analysis. A data
191
matrix of three stages from three genotypes was generated. Totally 19,269 to 20,928
192
genes were detected as expressed in these tissues at different stages representing
193
34.53% to 37.50% of total annotated genes of the Nipponbare reference genome
194
(Supplemental Table S1) (Kawahara et al., 2013). A total of 8,339, 6,278 and 530
195
genes were differentially expressed in BL5+ compared with BL at MMC, MEI and
196
AME, respectively, including 4,234, 2,430 and 410 genes that were up-regulated at
197
the three stages and 4,105, 3,848 and 120 genes that were down-regulated
198
(Supplemental Table S2; Supplemental Dataset 1). Among the differentially expressed
199
genes (DEGs), 2,067 were up-regulated and 3,325 were down-regulated at both MMC
200
and MEI (Fig. 2A). These 5,392 common DEGs accounted for 64.66% and 85.89% of
201
total DEGs at MMC and MEI, respectively, indicating that MMC and MEI involved
202
substantially overlapping cellular and molecular responses.
203
An overview of the differentially induced processes in BL5+ revealed by the
204
transcriptomes. We performed MapMan and Gene Ontology (GO) enrichment
205
analyses to classify the DEGs. MapMan is an effective tool to analyze metabolic
206
pathways and other biological processes of the transcriptome systematically (Thimm
207
et al., 2004; Ramsak et al., 2014). To get a global view of the 8,339 DEGs in BL5+ at
208
MMC, we used the “Metabolism overview” and “Cellular response” of MapMan to
209
outline the transcriptional changes of genes with putative functions in metabolism and
210
cellular response (Fig. 2B-C).
211
In “Metabolism overview”, primary metabolic processes, including cell wall (354
212
genes), amino acid (226 genes), lipids (316 genes), nucleotide (81 genes) and major
213
carbohydrate (128 genes) metabolism, accounted for 13.25% of total DEGs. The cell
9
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
214
wall localization of ORF5+, which was previously hypothesized to initiate the
215
premature PCD (Yang et al., 2012), led us to focus our attention to the cell wall
216
metabolic genes. We found that 187 (128 up-regulated and 59 down-regulated) of the
217
354 genes were involved in cell wall degradation and modification, which may affect
218
the cell wall structures in one way or another. The secondary metabolism processes
219
including terpenes (64 genes), flavonoids (105 genes), and phenylpropanoids (120
220
genes) metabolism, which are important in plant stress response (Dixon and Paiva,
221
1995; Winkelshirley, 2002; Tholl, 2006), constituted 3.47% of all DEGs. In the term
10
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
222
of “Cellular response”, 8.65% (721 genes) of DEGs were responsive to biotic and
223
abiotic stress; 1.94% (162 genes) of DEGs were related to redox, a phenomenon
224
usually triggered by stress; 6.67% (667 genes) of DEGs functioned in developmental
225
processes.
226
GO analysis for up-regulated genes revealed that three GO terms related to cell
227
wall metabolism, seven GO terms involved in stress or stimulus response, and eleven
228
GO terms associated with gene expression, protein synthesis and translation were
229
significantly enriched (FDR < 0.005) (Fig. 3).
230
The change in cell wall structure by cell wall modification genes may cause cell
231
wall integrity damage, which was similar to the situation of pathogen or pest attack.
232
We thus explored the “Biotic stress” in BL5+ at MMC and found that DEGs in
233
various functional categories of biotic stress were enriched including R genes,
234
pathogen-related proteins, signaling, hormone signaling, proteolysis, ROS-regulation
235
and biotic stress-responsive transcription factors (Supplemental Fig. S3A).
236
Thus, both of the MapMan and GO results indicated that a severe stress response
237
including both biotic and abiotic stresses were induced in BL5+ at MMC, which may
238
be caused by the damage in cell wall integrity, even though no abnormalities were
239
observed at this stage (Fig. 1F).
240
At MEI, 163 of the 300 DEGs in “cell wall” were related to the cell wall
241
degradation and modification including 102 up-regulated and 61 down-regulated ones
242
(Fig. 2B-C). Cell wall-related GO terms such as “glucan metabolic process”,
243
“polysaccharide metabolic process” and “polysaccharide biosynthetic process” were
244
enriched (Fig. 3). The continuous up-regulation of cell wall degrading and modifying
245
genes at MMC and MEI might be related to the observed cell wall abnormalities such
246
as vacuolization and degradation at MEI (Fig. 1G-H). In addition, 599 DEGs (9.54%
247
of total DEGs) in “biotic stress” and “abiotic stress” and genes in various functional
248
categories of biotic stress were identified (Fig. 2B, Supplemental Fig. S3B),
249
suggesting that the stress response also continued at MEI.
250
By the AME stage, DEG numbers in all the metabolic processes and cellular
251
responses were largely reduced compared to those at MMC and MEI (Fig. 2B). The
11
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
252
ovule had aborted with the accumulation of unknown substances. In contrast, however,
253
a GO term “cellulose biosynthetic process” was significantly enriched (Fig. 3),
254
indicating misregulation of cellulose biosynthesis in BL5+.
12
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
255
Large-scale transcriptional changes of cell wall modifying and degrading
256
genes in BL5+ at MMC and MEI. The MapMan and GO results showed that cell
257
wall metabolism especially cell wall modification and degradation were active in
258
BL5+ (Fig. 2). Cell wall modifying and degrading genes, also called cell wall
259
remodeling genes, affect cell growth by changing the extensibility and
260
physiochemical properties of the cell wall (Cosgrove, 1999; Yu et al., 2011; Gou et al.,
261
2012; Hepler et al., 2013; Xiao et al., 2014). We analyzed the expression of genes
262
from
263
endotransglucosylase/hydrolases (XTHs), pectin methyl esterases (PMEs), pectin
264
acetylesterases (PAEs), polygalacturonases (PGases), glycoside hydrolase family 9
265
(GH9) and arabinogalactan proteins (AGPs), which affect cell wall structure and
266
extension by enzymatic or non-enzymatic ways (Bosch et al., 2005; Coimbra et al.,
267
2008; Maris et al., 2009; Gou et al., 2012; Che et al., 2016). We found that 95, 85 and
268
23 genes from these families were differentially expressed at MMC, MEI, and AME,
269
respectively, of which 73 genes were differentially expressed at both MMC and MEI,
270
including 59 up-regulated and 14 down-regulated (Fig. 4A-B). The 59 up-regulated
271
genes consisted of 13 EXPs, 7 XTHs, 5 PMEs, 4 PAEs, 8 PGases, 7 GH9s and 15
272
AGPs. OsEXP4 was elevated in BL5+ at MMC and MEI. In rice, overexpression of
273
OsEXP4 results in increased cell length, while repression of OsEXP4 reduces cell
274
length (Choi et al., 2003). Three up-regulated XTHs (Os06g48160, Os03g01800 and
275
Os10g39840) have been characterized to utilize xyloglucan, one of the cell wall
276
components, as substrate (Hara et al., 2014). OsGLU1, a GH9 member, which
277
regulates cell elongation in rice (Zhou et al., 2006; Zhang et al., 2012), was found
278
up-regulated as well. The up-regulation of genes involved in cell wall degradation and
279
cell size regulation was in accordance with cell wall abnormalities in BL5+ (Fig.
280
1G-H). Thus, we inferred that the up-regulation of the cell wall remodeling genes
281
induced by ORF5+ in BL5+ led to cell structure change, damaging the cell wall
282
integrity.
seven
main
enriched
families:
expansins
(EXPs),
xyloglucan
283
Cellulose deposition in BL5+. In BL5+, unknown substances were accumulated
284
in the aborted ovule at AME (Fig. 1I-J). The enrichment of GO term “cellulose
13
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
285
biosynthetic process” suggested a misregulation of cellulose biosynthesis and that the
286
deposited substances in the aborted ovule might over-accumulate cellulose. To
287
validate this, we used Pontamine fast scarlet 4B (S4B) and calcofluor white to stain
288
cellulose (Anderson et al., 2010; Thomas et al., 2012), and observed strong
289
fluorescence signals in BL5+ at the site where the substances accumulated, indicating
290
that cellulose was indeed in large quantity (Fig. 5B, E), which was also supported by
291
the much higher cellulose content in panicles of BL5+ than in BL (Fig. 5G). In
292
contrast, normal mature embryo-sac structures were easily identified in the ovule of
293
BL and BL3+5+, where low fluorescence intensity was detected (Fig. 5A, C, D, and
294
F).
295
Cellulose is a major cell wall polysaccharide composed of unbranched β-1,
296
4-linked glucan chains. In rice, OsMYB46 and OsSWNs are secondary cell wall
14
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
297
biosynthetic regulators. OsSWN1 acts on the upstream of OsMYB46 by binding to the
298
SNBE element in its promoter region. Overexpression of OsSWN1 or OsMYB46 in
299
Arabidopsis results in the ectopic deposition of cellulose, xylan and lignin (Zhong et
300
al., 2011). In BL5+, OsSWN1, OsSWN4, OsSWN6 and OsMYB46 were up-regulated
301
at MMC and MEI. Three cellulose synthases, OsCESA4, OsCESA7 and OsCESA9,
302
which are regarded as subunits of cellulose synthase complex (Tanaka, 2003), were
303
continuously up-regulated from MMC to AME. In addition, expression of five
304
cellulose synthase-like genes (OsCslA1, OsCslA3, OsCslA5, OsCslA9 and OsCslF6)
305
and two brittle culm genes (BC1 and BC3) were also elevated at MMC or MEI (Fig.
306
4C). Upstream transcription factors such as OsSWN1, OsSWN6 and OsMYB46 had
307
peak expression at MEI while their downstream genes such as OsCESAs and two
308
brittle culm genes had highest expression at AME (Supplemental Fig. S4), in
309
agreement with the over-accumulation of cellulose at AME (Fig. 5B, E). These results
310
showed that the cellulose biosynthetic pathway was highly activated in BL5+
311
resulting in the accumulation of cellulose in the aborted embryo-sac.
312
Callose deposition in BL5+. Callose is a dynamic component of plant cell wall,
313
which plays important roles in megasporogenesis. The uneven distribution of the
314
callose wall around the megaspores decides the polarization of megaspores, the
15
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
315
formation of a functional megaspore and the abortion of the other three non-functional
316
megaspores (Ünal et al., 2013). Since many genes encoding β-1,3-glucanases, which
317
have putative functions in callose metabolism, were differentially expressed in BL5+
318
at MMC and MEI (Supplemental Fig. S5), we investigated the dynamic callose
319
distributions in ovule of BL, BL5+ and BL3+5+. During meiosis, callose
320
preferentially accumulated at the micropylar end of megaspores. Two callose bands
321
were observed in all three genotypes at the dyad stage (Fig. 6A-F), and at tetrad stage
322
at least three callose bands could be recognized also in all three genotypes although
323
slightly different in distribution patterns (Fig. 6G-L). This apparent normal callose
324
distribution in megaspores of BL5+ indicated likely normal meiosis. Unlike BL and
325
BL3+5+, however, weak callose deposition was detected in nucellus cells at the
326
tetrad stage in BL5+ (Fig. 6I). At the functional megaspore (FM) stage, callose
327
accumulated in the three degenerated gametes but not in the FM (Fig. 6M-R). The
328
survived FM subsequently developed to embryo-sac (Fig. 6S, T, W, X). However, the
16
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
329
ovule of BL5+ displayed irregular callose deposition around the FM and in nucellus
330
cells (Fig. 6O, P), and finally, the aborted embryo-sac was stuffed with large amount
331
of callose (Fig. 6U, V). Thus, ORF5+ resulted in a misregulation of callose deposition
332
during megasporogenesis such that callose began to accumulate abnormally from MEI
333
and was deposited in large quantity at AME in the ovule of BL5+.
334
ER stress and premature PCD induced in BL5+. Under stress conditions, the
335
number of misfolded proteins will increase and may exceed the capacity of the ER
336
quality-control system, thus inducing ER stress (Howell, 2013; Wan and Jiang, 2016).
337
In BL5+, 78.95% genes in “RNA transcription”, 66.23% genes in “RNA processing”,
338
57.69% genes in “protein synthesis initiation”, 58.33% genes in “protein synthesis
339
elongation”, 82.46% genes in “ribosomal protein synthesis” and 60.91% genes in
340
“ribosome biogenesis” were up-regulated at MMC (Supplemental Fig. S6), suggesting
341
a greatly increased level of protein synthesis, which may increase the protein folding
342
burden on ER. To detect whether ER stress occurred in BL5+ at MMC, we examined
343
the expression of ER stress responsive genes in BL5+ and found that 10 such genes
344
were significantly up-regulated including OsbZIP50, which is a key ER stress
345
regulator in rice (Fig. 7A). The mRNA of OsbZIP50 was spliced when ER stress
346
occurred (Hayashi et al., 2012). We then detected the unconventional splicing of
347
OsbZIP50 mRNA in different materials and found that OsbZIP50 mRNA was spliced
348
in BL5+ at MMC, but not in BL or BL3+5+ (Fig. 7B). The spliced OsbZIP50 mRNA
349
encodes a transcription factor OsbZIP50-S, which binds to the pUPRE-II cis-element
350
in the promoter regions of OsBIP4, ORF3, OsEBS and OsPDIL2-3 (Hayashi et al.,
351
2013; Takahashi et al., 2014; Wakasa et al., 2014). Accordingly, expression of these
352
four genes was elevated (Fig. 7A). Some other genes including Fes1-like, CRT2-2,
353
CRT1 and SAR1B-like, which were induced in ER stress, were also up-regulated.
354
These results showed that ER stress occurred in BL5+ at MMC relative to BL and
355
BL3+5+. In response to ER stress, genes involved in protein modification (303
356
up-regulated and 358 down-regulated) and targeting (70 up-regulated and 53
357
down-regulated) were differentially expressed presumably to help protein processing
358
and transportation. In addition, 793 DEGs (339 up-regulated and 454 down-regulated)
17
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
359
involved in “protein degradation” were found, likely as remedy to degrade the
360
misfolded or unfolded proteins (Supplemental Fig. S6). The splicing of OsbZIP50
361
mRNA and up-regulation of ER stress responsive genes were also detected at MEI
362
and AME (Fig. 7A-B), indicating that ER stress proceeded continuously in BL5+.
363
The unresolved ER stress could induce premature programmed cell death (PCD)
364
(Cai et al., 2014). In BL5+, PCD signals were detected from the meiosis stage (Yang
365
et al., 2012), corresponding to the MEI stage in this study. At MEI, we detected
366
up-regulation of two aspartic protease-encoding genes, OsAP25 and OsAP37 (Fig. 7A,
367
C), both of which participate in the tapetal PCD process and induce PCD in yeast and
368
plants (Niu et al., 2013). Overexpression of OsAP25 or OsAP37 in plants results in
369
cell death, which is suppressed by aspartic protease-specific inhibitor pepstatin A.
370
Transformation of OsAP25 or OsAP37 into a PCD-deficient yeast strain restores the
371
PCD ability of the strain. OsAP25 showed the highest expression at MEI (Fig. 7C),
18
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
372
consistent with the observation of PCD signals at this stage. In tapetal PCD process,
373
transcription factor EAT1 directly regulates the expression of OsAP25 and OsAP37
374
(Niu et al., 2013). However, EAT1 was down-regulated at MEI, indicating that some
375
other regulators might have replaced EAT1 to activate OsAP25 and OsAP37. TDR and
376
TIP2, which work upstream of OsAP25 and OsAP37 in pollen development (Niu et al.,
377
2013), were up-regulated in BL5+ at MMC and MEI (Fig. 7A, D). The coordinated
378
up-regulation of TDR, TIP2, OsAP25 and OsAP37 suggested that the ovule and
379
tapetum may share some common genes/pathways in PCD. Taken together, an
380
OsbZIP50-dependent ER stress pathway and a premature PCD pathway involving the
381
tapetal PCD genes were activated in BL5+.
382
These results suggested that the ORF5+-induced up-regulation of cell wall
383
degradation and modification genes in BL5+ caused cell wall damage, which induced
384
both biotic and abiotic stresses. Lasting stress responses induced ER stress and
385
unresolved ER stress led to premature PCD eventually resulting in the ovule abortion.
386
Processes detected in the BL3+5+ /BL comparison.
387
BL3+5+ carrying both the killer ORF5+ and the protector ORF3+ showed no
388
phenotypic difference from the wild type BL with respect to fertility (Supplemental
389
Fig. S1). However, 3,986 DEGs were still identified between BL3+5+ and BL at
390
MMC (Supplemental Table S2; Supplemental Dataset 2), of which 3,843 DEGs were
391
shared with the BL5+/BL comparison at this stage, indicating that ORF5+ induced
392
similar processes in BL3+5+ (Fig. 8A). As expected, the process of “cell wall” was
393
enriched with 217 DEGs (Fig. 8B), of which 64 (55 up-regulated and 9
394
down-regulated) were cell wall modifying and degrading genes from the seven main
395
families (Fig. 4A-B). GO terms “glucan metabolic process” and “polysaccharide
396
metabolic process” were significantly enriched in up-regulated genes (Fig. 8C). Terms
397
“biotic stress” and “abiotic stress” were also enriched involving 339 DEGs. However,
398
the number of DEGs in each of the enriched functional categories was smaller than
399
that in the BL5+/BL comparison (Fig. 4A-B). Moreover, the splicing of OsbZIP50
400
mRNA and the up-regulation of its downstream genes were not detected (Fig. 7A-B).
401
These results suggested that ORF5+ induced similar processes in BL3+5+ at MMC
19
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
402
especially with respect to cell wall integrity damage, but to a much lesser extent,
403
which did not trigger ER stress.
404
At MEI, the difference between BL3+5+ and BL was greatly narrowed that only
405
749 DEGs were identified, compared with 6,278 genes in the BL5+/BL comparison
406
(Supplemental Table S2; Supplemental Dataset 2). Thus numbers of DEGs especially
407
those in cell wall, biotic stress and abiotic stress were greatly reduced (Fig. 8B). Only
408
7 DEGs from the seven cell wall families were found (Fig. 4A-B), suggesting that cell
409
wall integrity damage was suppressed. The cell wall compensation pathways such as
410
cellulose biosynthesis and callose deposition were not activated (Fig 4C; Fig. 6K, Q),
411
and ER stress and PCD did not occur (Fig. 7). As a result, the cell wall maintained
20
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
412
integrity and the ovule meiosis was undisturbed (Fig. 1L-M).
413
At AME, the number of DEGs was further reduced to 370 in BL3+5+ compared
414
with BL (Supplemental Table S2; Supplemental Dataset 2). These results indicated
415
that ORF5+ induced similar but weaker damage processes in BL3+5+ as in BL5+ at
416
MMC, but these processes were soon suppressed by ORF3+. Consequently, the
417
embryo-sac developed normally producing fertile female gametes (Fig. 1O).
418
419
21
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
420
DISCUSSION
421
The killer-protector system of S5 is composed of three proteins, cell wall protein
422
ORF5+, transmembrane protein ORF4+, and ER resident protein ORF3+. Based on
423
the comparative transcriptome results of BL5+ and BL3+5+, we proposed a model to
424
summarize the processes underlying the S5 system (Fig. 9).
425
In BL5+, many cell wall modifying and degrading genes in the families EXPs,
426
XTHs, PMEs, PAEs, PGases, GH9, and AGPs were up-regulated at MMC and MEI,
427
and many genes of these families play important roles in cell elongation or cell
428
expansion (Zhou et al., 2006; Chen and Ye, 2007; Yang et al., 2007; Coimbra et al.,
429
2008; Maris et al., 2009; Zhang et al., 2010; Yu et al., 2011; Gou et al., 2012; Zhang et
430
al., 2012; Ma et al., 2013; Miedes et al., 2013; Hara et al., 2014; Xiao et al., 2014;
431
Che et al., 2016). It has been suggested that post-synthetic modifications of the cell
432
wall by cell wall modifying genes may affect cell wall integrity (CWI) (Pogorelko et
433
al., 2013). Thus, large-scale up-regulation of cell wall remodeling genes induced by
434
ORF5+ may lead to changes of cell wall structures, which affect CWI thus inducing
435
stress response.
436
ORF5+ encodes an extracellular aspartic protease. In yeast, extracellular aspartic
437
proteases yapsins are required for the CWI maintenance (Krysan et al., 2005; Guan et
438
al., 2012). Two plant aspartic proteases A36 and A39 participate in cell wall
439
remodeling in pollen tube (Gao et al., 2017). The study in Candida albicans shows
440
that secreted aspartic proteinases use a set of cell wall proteins as substrates (Schild et
441
al., 2011). We speculated that one or more substrates of ORF5+ existed in the rice cell
442
wall, which induced cell wall damage after activated by ORF5+. In yeast, CWI
443
damage triggers unfolded protein response (UPR), a homeostatic response to alleviate
444
ER stress, which in turn affects the CWI (Krysan, 2009; Scrimale et al., 2009). In
445
plants, pathogen invasion or pest attack can induce CWI damage and subsequent
446
stress response (Bellincampi et al., 2014; Pogorelko et al., 2013). CWI damage
447
eventually leads to PCD (Paparella et al., 2015). In BL5+, the functional categories
448
responsive to pathogen invasion or pest attack were enriched, and ER stress was
449
accordingly induced presumably by CWI impairment, which in turn exacerbated the
22
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
450
cell wall damage and its subsequent stress responses, thus finally leading to PCD. To
451
alleviate the adverse effects caused by CWI damage, cells usually trigger a cell wall
452
compensatory pathway to strengthen the cell wall (Hamann, 2012). For example,
453
disruption by aspartic protease Yps7p in Pichia pastoris reduces chitin content in the
454
lateral cell wall, but the cell thickens the inner cell wall for compensation (Guan et al.,
455
2012). Similarly in BL5+, key genes in secondary cell wall biosynthesis were
456
significantly up-regulated resulting in the cellulose accumulation to repair cell wall.
457
Callose, a polysaccharide usually accumulated under stress conditions to provide
23
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
458
physical barrier to the adverse environments (Pirselova and Matusikova, 2012), was
459
deposited in large quantity. Despite other roles of callose in ovule development, the
460
large amount deposition of callose in BL5+ was most likely to strengthen cell wall.
461
ORF4+ acted as a partner of ORF5+ with a transmembrane localization. We
462
speculated that ORF4+ functioned as a sensor of CWI damage. In plants, a
463
well-studied cell wall sensor is AtWAK1, a wall-associated kinase, which resides in
464
the plasma membrane and responds to oligogalacturonides derived from the
465
hydrolysis of cell wall component (Brutus et al., 2010). Xa4, a wall-associated kinase
466
in rice, contributes to disease resistance by strengthening the cell wall via promoting
467
cellulose synthesis and suppressing cell wall loosening (Hu et al., 2017). Unlike
468
wall-associated kinase, no extracellular binding domain or inner kinase domain was
469
found in ORF4+, thus there may exist a co-receptor that interacts with ORF4+ in the
470
plasma membrane and/or a downstream signal protein that interacts with plasma
471
membrane receptor to transmit signals.
472
Based on these analyses, it was deduced that ORF5+ damaged CWI by cleaving
473
its substrates leading to up-regulation of the cell wall modifying and degrading genes.
474
ORF4+ sensed the CWI impairment and induced biotic and abiotic stress responses,
475
consequently induced ER stress due to the accumulation of drastically increased
476
misfolded proteins in ER. The occurrence of ER stress in turn deteriorated the CWI
477
damage and led to a more serious stress response. Finally, the unresolved cell wall
478
damage, stress response and ER stress triggered PCD. Meanwhile, the cellulose and
479
callose accumulated in ovule as the cells’ unsuccessful remedy to repair the damaged
480
cell wall.
481
BL3+5+ showed no difference with BL with respect to ovule development and
482
fertility, but a number of cell wall modifying and degrading genes were still
483
up-regulated in BL3+5+ at MMC, which may induce CWI damage. However,
484
transcriptional change of these genes was suppressed at MEI and AME. Thus, the
485
subsequent negative effects caused by CWI damage including ER stress, PCD,
486
cellulose and callose deposition were prevented in BL3+5+. ORF3+ encodes a heat
487
shock protein 70 chaperone, which is a downstream target of OsbZIP50s and induced
24
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
488
by ER stress. ORF3+ is believed to help protein folding in ER to resolve ER stress
489
like OsBIP1-OsBIP5 in the HSP70 gene family (Yang et al., 2012; Hayashi et al.,
490
2013; Wakasa et al., 2014). BIP protein alleviates Cd2+ stress-induced PCD (Xu et al.,
491
2013). The tobacco plants with overexpression of BIP show delayed leaf senescence
492
(Carvalho et al., 2014). In BL3+5+, ORF3+ suppressed the ORF5+-induced ER
493
stress and PCD. It was speculated that one role of ORF3+ was alleviating ER stress
494
thus not to induce premature PCD. The chaperone activity of ORF3+ may also help
495
the folding of the signal proteins downstream of ORF5+ and ORF4+ so that the stress
496
response may be suppressed at MEI.
497
In conclusion, we revealed the processes underlying a reproductive barrier
498
composed of ORF3, ORF4, and ORF5 at the transcriptomic level and proposed a
499
mechanistic model based on the results. Nevertheless, further studies are needed to
500
support and enrich the model.
501
502
MATERIALS AND METHODS
503
Sample preparation and library construction
504
Balilla was a typical japonica rice variety. The transgenic plant BalillaORF5+
505
was generated by introducing ORF5+ from an indica variety Nanjing 11 into Balilla
506
under its native promoter (Chen et al., 2008). The plant BalillaORF3+ORF5+ was
507
isolated from the hybrid offspring between BalillaORF5+ and BalillaORF3+, which
508
contained a transformed ORF3+ from Nanjing 11 under its native promoter (Yang et
509
al., 2012). All the rice plants were grown in the fields of the experimental station of
510
Huazhong Agricultural University, Wuhan, China. We determined the relationship
511
between the floret length and stage of ovule development by the paraffin section
512
observation of florets from the variety Balilla. The pistils from florets of Balilla,
513
BalillaORF5+ and BalillaORF3+ORF5+ in length ranges of 3.8-4.2 mm, 5.5-6.0 mm,
514
and 7.0-7.5 mm were collected as MMC, MEI and AME stage samples with two
515
biological replicates. RNA was extracted using TRIzol reagent (Invitrogen) according
516
to the manufacture’s manual. RNA yields and quality were measured using Nanodrop
517
2000 (Thermo Scientific), and further quantified using Qubit2.0 Fluorometer
25
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
518
(Invitrogen). RNA integrity was evaluated using Experion RNA Analysis Kits
519
(Bio-rad) and Experion Automated Electrophoresis Station (Bio-rad). Each library
520
was constructed using 3 μg total RNA using the TruSeq RNA Library Preparation Kit
521
v2 (Illumina). The libraries were sequenced using Illumina HiSeq 2000 sequencing
522
platform at National Key Laboratory for Crop Genetic Improvement in Huazhong
523
Agricultural University, Wuhan, China.
524
Data processing and DEGs identification
525
The data of the two replicates of all samples were collected, and analyzed
526
following the workflow described previously (Trapnell et al., 2012). In brief, the raw
527
sequencing reads were mapped to the rice reference genome (MSU7.0,
528
http://rice.plantbiology.msu.edu/index.shtml) using TopHat, then the transcripts were
529
assembled and quantified using Cufflinks, and finally, differentially expressed genes
530
were analyzed using Cuffdiff. The raw data files can be found in the NCBI database
531
under the accession numbers GSE95200. Genes with ≥ 2-fold expression
532
up-regulation or down-regulation and adjusted p-value ≤ 0.05 (counted using Cuffdiff)
533
were regarded as up-regulated or down-regulated genes.
534
GO and MapMan analysis
535
Gene ontology (GO) enrichment was performed in AgriGO, a web-based tool for
536
gene ontology analysis (Du et al., 2010) (http://bioinfo.cau.edu.cn/agriGO/index.php).
537
The species “Oryza sativa” were chosen. GO terms in biological process groups with
538
FDR < 0.005 were identified and analyzed further. The MapMan software version
539
3.5.1.R2 and pathways were downloaded from MapMen Site of analysis
540
(http://mapman.gabipd.org/web/guest/mapmanstore). The mapping information of
541
rice genes (osa_MSU_v7_2015-09-08_mapping.txt.tar.gz) that can be imported to the
542
MapMan
543
(http://www.gomapman.org/export/current/mapman) (Ramsak et al., 2014).
544
In situ hybridization
software
was
downloaded
from
GoMapMan
545
The methods for preparation of paraffin embedded sections followed the
546
previously described protocol (Weng et al., 2014). Primer pairs (listed in
547
Supplemental Table S4) ORF3insituF and ORF3insituR, ORF4insituF and
26
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
548
ORF4insituR were used to prepare probes for ORF3 and ORF4, respectively. Products
549
amplified from ORF3+ or ORF4+ cDNA were ligated to pGEM-T vector (Promega).
550
The sense probe was transcribed with T7 RNA polymerase, and the antisense probe
551
was transcribed with SP6 RNA polymerase. The probes were labeled with
552
digoxigenin (Roche). Hybridization and detection were performed as described
553
previously (De Block and Debrouwer, 1993).
554
PCR and RT-qPCR
555
Total RNA of pistils were isolated with TRIzol reagent (Invitrogen) and treated
556
with RNase-free DNaseI (Invitrogen) for 20 min at room temperature to digest the
557
contaminating DNA. The first-strand cDNA was synthesized using SSIII reverse
558
transcriptase (Invitrogen) with Oligo(dT)15 Primer (Promega) following the
559
manufacturers’ instruction. For amplification of OsbZIP50s, specific primers around
560
the splice site were used. PCR conditions and PCR products detection were performed
561
as described before (Yang et al., 2012). For quantitative real-time PCR, we carried out
562
the reaction in a total volume of 10 μl containing 5 μl FastStart Universal SYBR
563
Green Master (Rox) (Roche), 1 μl cDNA sample, 3 μM each primer on ABI ViiA™7
564
system. The primers for PCR and RT-qPCR are listed in Supplemental Table S4. The
565
gene profilin (Os06g05880) was used as the internal control in RT-qPCR (Yang et al.,
566
2012).
567
Histological staining
568
The fresh florets were fixed with FAA solution (50% ethanol, 5% acetic acid, 3.7%
569
formaldehyde) at 4°C overnight, dehydrated with ethanol in a series of concentrations
570
(50%, 70%, 85%, 95%, 100%), infiltrated with xylene and finally embedded in
571
paraffin. The materials were cut into 10 μm-thick sections and then washed in a
572
xylene/ethanol series (0–50% ethanol). For callose staining, the rehydrated sections
573
were stained with 0.1% aniline blue solution for 60 min. For cellulose staining, the
574
rehydrated sections were stained with 0.01% Pontamine Fast Scarlet 4B solution for
575
20 min or 0.1% calcofluor white (Sigma) for 5 min. The stained sections were
576
scanned under fluorescence microscopy. The florets for observation of ovule
577
development process were stained with ehrlich haematoxylin. After fixation, the
27
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
578
materials were rehydrated, infiltrated with ehrlich haematoxylin for four days, then
579
dehydrated with ethanol, infiltrated with xylene and embedded in paraffin. The 10
580
μm-thick sections were dewaxed with xylene and sealed with Canada balsam (Sigma).
581
The slides were scanned under light microscopy.
582
Cellulose content determination
583
The cellulose content was determined as described previously (Guo et al., 2014).
584
In brief, the materials were suspended with potassium phosphate buffer (pH 7.0),
585
chloroform-methanol (1:1, v/v), dimethylsulphoxide (DMSO)-water (9:1, v/v), 0.5%
586
(w/v) ammonium oxalate, 4 M KOH containing 1.0 mg/mL sodium borohydride in
587
turn. The remaining pellet was collected and extracted further with H2SO4 (67%, v/v)
588
and the supernatants were collected for the determination of cellulose.
589
590
SUPPLEMENTAL DATA
591
Supplemental Figure S1. The fertility of BL, BL5+, and BL3+5+.
592
Supplemental Figure S2. RNA in situ hybridization of ORF3+ and ORF4+.
593
Supplemental Figure S3. DEGs in “Biotic stress” in BL5+ compared with BL at
594
MMC (A) and MEI (B).
595
Supplemental Figure S4. qPCR verification for expression of the cellulose
596
biosynthetic genes in pistils of BL, BL5+, and BL3+5+ at MMC, MEI, and AME,
597
respectively.
598
Supplemental Figure S5. Expression change of genes involved in callose
599
metabolism.
600
Supplemental Figure S6. The number of DEGs involved in RNA transcription,
601
protein synthesis and processing.
602
Supplemental Table S1. The number of expressed genes in BL, BL5+, and BL3+5+
603
at different stages.
604
Supplemental Table S2. The number of differentially expressed genes identified in
605
different comparisons.
606
Supplemental Table S3. Full list of significantly enriched GO terms (FDR < 0.005)
607
for DEGs in BL5+ vs. BL.
28
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
608
Supplemental Table S4. Full list of significantly enriched GO terms (FDR < 0.005)
609
for DEGs in BL3+5+ vs. BL.
610
Supplemental Table S5. The list of cell wall modifying or degrading genes and their
611
expression changes based on log2 fold.
612
Supplemental Table S6. The primers used in this study.
613
Supplemental Dataset 1. The differentially expressed genes in BL5+ compared with
614
BL.
615
Supplemental Dataset 2. The differentially expressed genes in BL3+5+ compared
616
with BL.
617
618
ACKNOWLEDGMENTS
619
We thank Yihua Zhou from Institute of Genetics and Developmental Biology for
620
kindly providing the Pontamine Fast Scarlet 4B solution.
621
29
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
622
623
FIGURE LEGENDS
624
Figure 1. The ovule development process of BL, BL5+, and BL3+5+.
625
(A-E) Ovule development of BL (gametes fertile). (F-J) Ovule development of BL5+
626
(gametes sterile). (K-O) Ovule development of BL3+5+ (gametes fertile). (A, F, K)
627
The megaspore mother cell stage. (B, G, L) The dyad stage. (C, H, M) The tetrad
628
stage. (D, I, N) The functional megaspore stage. (E, J, O) The mature embryo sac.
629
MMC, megaspore mother cell; Dy, dyad; Te, tetrad; FM, functional megaspore; DM,
630
degenerated megaspore; AFM, aborted functional megaspore; ANC, abnormal
631
nucellus cells; ES, embryo sac; AES, aborted embryo sac. Bars = 30 μm. The grey
632
bars at the top indicate the corresponding stage in transcriptome data. MMC indicated
633
the megaspore mother cell stage. MEI indicates the meiosis stage. AME indicates the
634
stage after meiosis.
635
636
Figure 2. Analysis of genes differentially expressed in BL5+ compared to BL.
637
(A) Numbers of the up- and down-regulated DEGs at MMC and MEI. (B) Numbers of
638
DEGs in different MapMan functional categories at MMC, MEI and AME,
639
respectively. (C) The heat map of “Metabolism overview” and “Cellular response” in
640
BL5+ at MMC based on the MapMan results. Each small square represents a gene
641
differentially expressed in BL5+ vs. BL. The color of the square represents the level
642
of up-regulation (red) or down-regulation (green) based on the log2 fold change of the
643
DEGs.
644
645
Figure 3. Partial significantly (FDR < 0.005) enriched GO terms for up-regulated
646
genes in BL5+ compared to BL.
647
The color in each cell represents the significance of enrichment based on the FDR
648
value. The cells without color indicate not significantly enriched. The full list of GO
649
terms is given in Supplemental Table S3.
650
651
Figure 4. Differential regulation of cell wall genes in BL5+ and BL3+5+ compared
30
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
652
with BL.
653
(A) The number of differentially expressed genes involved in cell wall modification
654
and degradation in BL5+ and BL3+5+. The X-axis indicates the gene numbers. The
655
Y-axis indicates the gene families at different stages. (B) Heat map of expression
656
change of genes involved in cell wall modification and degradation. The color in each
657
cell represents the level of expression change based on the log2 fold. The cell without
658
color indicates the gene was not differentially expressed. Full list of genes and their
659
expressions is given in Supplemental Table S5. (C) Expression change of cellulose
660
biosynthetic genes.
661
662
Figure 5. Cellulose deposition in the mature ovules of BL, BL5+, and BL3+5+.
663
(A-F) Cellulose deposition in BL (A, D), BL5+ (B, E) and BL3+5+ (C, F). (A-C)
664
S4B staining. (D-F) Calcofluor white staining. The strong red or blue fluorescence
665
indicates where the cellulose accumulated. Bars = 50 μm. (G) Cellulose content in the
666
panicles of BL and BL5+. The data are means ± SEM (n = 3). *** significant
667
difference at P < 0.001.
668
669
Figure 6. Callose deposition (green fluorescence) in the developing ovule of BL,
670
BL5+, and BL3+5+ by aniline blue staining.
671
(A-F) The dyad stage. The red arrowheads indicate the callose bands around the dyad.
672
(G-L) The tetrad stage. The red arrowheads indicate the callose bands around the
673
tetrad. (M-R) The functional megaspore stage. The red arrowheads indicate the
674
degenerated megaspores. (S-X) The mature embryo-sac. (I, O, U) The grey
675
arrowheads indicate abnormally deposited callose. Bars = 25 μm.
676
677
Figure 7. Differential regulation of ER stress and PCD genes.
678
(A) Log2 fold change of the expression level of ER stress and PCD genes in BL5+
679
against BL. The color in each cell represents the levels of expression change based on
680
the log2 fold. The cell without color indicates the gene was not differentially
681
expressed. (B) Splicing of OsbZIP50 mRNA in BL, BL5+ and BL3+5+ at each stage.
31
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
682
(C) qPCR verification for expression of tapetal PCD-related genes in BL and BL5+.
683
The X-axis represents different stages. The Y-axis represents the expression level
684
relative to profilin (Os06g05880). The data are means ± SEM (n = 3).
685
686
Figure 8. Analysis of genes differentially expressed in BL3+5+ compared to BL.
687
(A) Number of DEGs detected in the BL3+5+/BL and BL5+/BL comparisons at
688
different stages. The overlapping areas indicate the genes differentially expressed in
689
both BL3+5+ and BL5+. (B) The number of DEGs in different functional categories
690
of “Metabolism Overview” and “Cellular response” by MapMan at MMC, MEI and
691
AME, respectively. (C) Partial significantly (FDR < 0.005) enriched GO terms for
692
up-regulated genes in BL3+5+ compared with BL. The color in each cell represents
693
the significance of enrichment based on the FDR value. The cells without color
694
indicate not significantly enriched. The full list of GO terms is given in Supplemental
695
Table S4.
696
697
Fig. 9. A model for S5 locus-mediated ovule abortion.
698
32
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
699
700
33
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
Parsed Citations
Anderson CT, Carroll A, Akhmetova L, Somerville C (2010) Real-time imaging of cellulose reorientation during cell wall expansion
in Arabidopsis roots. Plant Physiol 152: 787-796
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Bellincampi D, Cervone F, Lionetti V (2014) Plant cell wall dynamics and wall-related susceptibility in plant-pathogen interactions.
Front Plant Sci 5: 228
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Bosch M, Ycheung A, Khepler P (2005) Pectin methylesterase, a regulator of pollen tube growth. Plant Physiol 138: 1334-1346
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Brutus A, Sicilia F, Macone A, Cervone F, De Lorenzo G (2010) A domain swap approach reveals a role of the plant wall-associated
kinase 1 (WAK1) as a receptor of oligogalacturonides. Proc Natl Acad Sci U S A 107: 9452-9457
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Cai YM, Yu J, Gallois P (2014) Endoplasmic reticulum stress-induced PCD and caspase-like activities involved. Front Plant Sci 5:
41
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Carvalho HH, Silva PA, Mendes GC, Brustolini OJ, Pimenta MR, Gouveia BC, Valente MA, Ramos HJ, Soares-Ramos JR, Fontes EP
(2014) The endoplasmic reticulum binding protein BiP displays dual function in modulating cell death events. Plant Physiol 164:
654-670
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Che J, Yamaji N, Shen RF, Ma JF (2016) An Al-inducible expansin gene, OsEXPA10 is involved in root cell elongation of rice. Plant
J 88: 132-142
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Chen J, Ding J, Ouyang Y, Du H, Yang J, Cheng K, Zhao J, Qiu S, Zhang X, Yao J, Liu K, Wang L, Xu C, Li X, Xue Y, Xia M, Ji Q, Lu J,
Xu M, Zhang Q (2008) A triallelic system of S5 is a major regulator of the reproductive barrier and compatibility of indica-japonica
hybrids in rice. Proc Natl Acad Sci U S A 105: 11436-11441
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Chen L, Ye D (2007) Roles of pectin methylesterases in pollen-tube growth. J Integr Plant Biol 49: 94-98
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Choi D, Lee Y, Cho HT, Kende H (2003) Regulation of expansin gene expression affects growth and development in transgenic
rice plants. Plant Cell 15: 1386-1398
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Coimbra S, Jones B, Pereira LG (2008) Arabinogalactan proteins (AGPs) related to pollen tube guidance into the embryo sac in
Arabidopsis. Plant Signal Behav 3: 455-456
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Coolen S, Proietti S, Hickman R, Davila Olivas NH, Huang P-P, Van Verk MC, Van Pelt JA, Wittenberg AHJ, De Vos M, Prins M, Van
Loon JJA, Aarts MGM, Dicke M, Pieterse CMJ, Van Wees SCM (2016) Transcriptome dynamics of Arabidopsis during sequential
biotic and abiotic stresses. Plant J 86: 249-267
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Cosgrove DJ (1999) Enzymes and other agents that enhance cell wall extensibility. Annu Rev Plant Physiol Plant Mol Biol 50: 391417
Pubmed: Author and Title
CrossRef: Author and Title
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
Google Scholar: Author Only Title Only Author and Title
Coyne J, Orr H (2004) Speciation. Sunderland, MA: Sinauer Associates
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
De Block M, Debrouwer D (1993) RNA-RNA in situ hybridization using digoxigenin-labelled probes: the use of high-molecularweight polyvinyl alcohol in the alkaline phosphatase indoxyl-nitroblue tetrazolium reaction. Anal Biochem 216: 86-89
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Digel B, Pankin A, von Korff M (2015) Global transcriptome profiling of developing leaf and shoot apices reveals distinct genetic
and environmental control of floral transition and inflorescence development in barley. Plant Cell 27: 2318-2334
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Dixon RA, Paiva NL (1995) Stress-induced phenylpropanoid metabolism. Plant Cell 7: 1085-1097
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Dobzhansky T (1937) Genetics and the origin of species. New York: Columbia University Press
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Du H, Ouyang Y, Zhang C, Zhang Q (2011) Complex evolution of S5, a major reproductive barrier regulator, in the cultivated rice
Oryza sativa and its wild relatives. New Phytol 191: 275-287
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Du Z, Zhou X, Ling Y, Zhang Z, Su Z (2010) agriGO: a GO analysis toolkit for the agricultural community. Nucleic Acids Res 38: W6470
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Gao H, Zhang Y, Wang W, Zhao K, Liu C, Bai L, Li R, Guo Y (2017) Two membrane-anchored aspartic proteases contribute to pollen
and ovule development. 173: 219-239
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Gou J, Mmiller L, Hou G, Yu X, Chen X, Liu C (2012) Acetylesterase-mediated deacetylation of pectin impairs cell elongation, pollen
germination, and plant reproduction. Plant Cell 24: 50-65
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Groszmann M, Gonzalez-Bayon R, Lyons RL, Greaves IK, Kazan K, Peacock WJ, Dennis ES (2015) Hormone-regulated defense
and stress response networks contribute to heterosis in Arabidopsis F1 hybrids. Proc Natl Acad Sci U S A 112: E6397-6406
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Guan B, Lei J, Su S, Chen F, Duan Z, Chen Y, Gong X, Li H, Jin J (2012) Absence of Yps7p, a putative glycosylphosphatidylinositollinked aspartyl protease in Pichia pastoris, results in aberrant cell wall composition and increased osmotic stress resistance.
FEMS Yeast Res 12: 969-979
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Guo K, Zou W, Feng Y, Zhang M, Zhang J, Tu F, Xie G, Wang L, Wang Y, Klie S, Persson S, Peng L (2014) An integrated genomic
and metabolomic framework for cell wall biology in rice. BMC Genomics 15: 596
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Hamann T (2012) Plant cell wall integrity maintenance as an essential component of biotic stress response mechanisms. Frontiers
in Plant Science 3: 77
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Hara Y, Yokoyama R, Osakabe K, Toki S, Nishitani K (2014) Function of xyloglucan endotransglucosylase/hydrolases in rice. Ann
Bot 114: 1309-1318
Pubmed: Author and Title
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Hayashi S, Takahashi H, Wakasa Y, Kawakatsu T, Takaiwa F (2013) Identification of a cis-element that mediates multiple pathways
of the endoplasmic reticulum stress response in rice. Plant J 74: 248-257
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Hayashi S, Wakasa Y, Takahashi H, Kawakatsu T, Takaiwa F (2012) Signal transduction by IRE1-mediated splicing of bZIP50 and
other stress sensors in the endoplasmic reticulum stress response of rice. Plant J 69: 946-956
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Hepler PK, Rounds CM, Winship LJ (2013) Control of cell wall extensibility during pollen tube growth. Mol Plant 6: 998-1017
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Howell SH (2013) Endoplasmic reticulum stress responses in plants. Annu Rev Plant Biol 64: 477-499
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Hu K, Cao J, Zhang J, Xia F, Ke Y, Zhang H, Xie W, Liu H, Cui Y, Cao Y, Sun X, Xiao J, Li X, Zhang Q, Wang S (2017) Improvement of
multiple agronomic traits by a disease resistance gene via cell wall reinforcement. Nat Plants 3: 17009
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Ikehashi H, Araki H (1986) Genetics of F1 sterility in remote crosses of rice. In: IRRI (eds) Rice genetics. IRRI, Manila, pp 119-130
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Kawahara Y, de la Bastide M, Hamilton JP, Kanamori H, McCombie WR, Ouyang S, Schwartz DC, Tanaka T, Wu J, Zhou S, Childs
KL, Davidson RM, Lin H, Quesada-Ocampo L, Vaillancourt B, Sakai H, Lee SS, Kim J, Numa H, Itoh T, Buell CR, Matsumoto T (2013)
Improvement of the Oryza sativa Nipponbare reference genome using next generation sequence and optical map data. Rice 6: 4
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Kourmpetli S, Lee K, Hemsley R, Rossignol P, Papageorgiou T, Drea S (2013) Bidirectional promoters in seed development and
related hormone/stress responses. BMC Plant Biol 13: 1-16
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Krysan DJ (2009) The cell wall and endoplasmic reticulum stress responses are coordinately regulated in Saccharomyces
cerevisiae. Commun Integr Biol 2: 233-235
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Krysan DJ, Ting EL, Abeijon C, Kroos L, Fuller RS (2005) Yapsins are a family of aspartyl proteases required for cell wall integrity in
Saccharomyces cerevisiae. Eukaryot Cell 4: 1364-1374
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Kubo T, Takashi T, Ashikari M, Yoshimura A, Kurata N (2016) Two tightly linked genes at the hsa1 locus cause both F1 and F2
hybrid sterility in rice. Mol Plant 9: 221-232
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Liu A, Zhang Q, Li H (1992) Location of a gene for wide-compatibility in the RFLP linkage map. Rice Genet Newslett 9: 134-136
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Liu K, Wang J, Li H, Xu C, Liu A, Li X, Zhang Q (1997) A genome-wide analysis of wide compatibility in rice and the precise location
of the S5 locus in the molecular map. Theor Appl Genet 95: 809-814
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Long Y, Zhao L, Niu B, Su J, Wu H, Chen Y, Zhang Q, Guo J, Zhuang C, Mei M, Xia J, Wang L, Wu H, Liu YG (2008) Hybrid male
sterility in rice controlled by interaction between divergent alleles of two adjacent genes. Proc Natl Acad Sci U S A 105: 1887118876
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Ma N, Wang Y, Qiu S, Kang Z, Che S, Wang G, Huang J (2013) Overexpression of OsEXPA8, a root-specific gene, improves rice
growth and root system architecture by facilitating cell extension. PLoS ONE 8: e75997
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Maris A, Suslov D, Fry SC, Verbelen JP, Vissenberg K (2009) Enzymic characterization of two recombinant xyloglucan
endotransglucosylase/hydrolase (XTH) proteins of Arabidopsis and their effect on root growth and cell wall extension. J Exp Bot
60: 3959-3972
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Miedes E, Suslov D, Vandenbussche F, Kenobi K, Ivakov A, Van Der Straeten D, Lorences EP, Mellerowicz EJ, Verbelen JP,
Vissenberg K (2013) Xyloglucan endotransglucosylase/hydrolase (XTH) overexpression affects growth and cell wall mechanics in
etiolated Arabidopsis hypocotyls. J Exp Bot 64: 2481-2497
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Mizuta Y, Harushima Y, Kurata N (2010) Rice pollen hybrid incompatibility caused by reciprocal gene loss of duplicated genes.
Proc Natl Acad Sci U S A 107: 20417-20422
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Muller HJ (1942) Isolating mechanisms, evolution, and temperature. Biol Symp 6: 71-125
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Niu N, Liang W, Yang X, Jin W, Wilson Z, Hu J, Zhang D (2013) EAT1 promotes tapetal cell death by regulating aspartic proteases
during male reproductive development in rice. Nat Commun 4: 1445
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Ouyang Y, Chen J, Ding J, Zhang Q (2009) Advances in the understanding of inter-subspecific hybrid sterility and widecompatibility in rice. Chin Sci Bull 54: 2332-2341
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Ouyang Y, Li G, Mi J, Xu C, Du H, Zhang C, Xie W, Li X, Xiao J, Song H, Zhang Q (2016) Origination and establishment of a trigenic
reproductive isolation system in rice. Mol Plant 9: 1542-1545
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Ouyang Y, Zhang Q (2013) Understanding reproductive isolation based on the rice model. Annu Rev Plant Biol 64: 111-135
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Paparella S, Tava A, Avato P, Biazzi E, Macovei A, Biggiogera M, Carbonera D, Balestrazzi A (2015) Cell wall integrity, genotoxic
injury and PCD dynamics in alfalfa saponin-treated white poplar cells highlight a complex link between molecule structure and
activity. Phytochemistry 111: 114-123
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Pirselova B, Matusikova I (2012) Callose: the plant cell wall polysaccharide with multiple biological functions. Acta Physiol Plant 35:
635-644
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Pogorelko G, Lionetti V, Bellincampi D, Zabotina O (2013) Cell wall integrity: targeted post-synthetic modifications to reveal its role
in plant growth and defense against pathogens. Plant Signal Behav 8: e25435
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Qiu SQ, Liu K, Jiang JX, Song X, Xu CG, Li XH, Zhang Q (2005) Delimitation of the rice wide compatibility gene S5n to a 40-kb DNA
fragment. Theor Appl Genet 111: 1080-1086
Pubmed: Author and Title
CrossRef: Author and Title
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
Google Scholar: Author Only Title Only Author and Title
Ramsak Z, Baebler S, Rotter A, Korbar M, Mozetic I, Usadel B, Gruden K (2014) GoMapMan: integration, consolidation and
visualization of plant gene annotations within the MapMan ontology. Nucleic Acids Res 42: D1167-1175
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Schild L, Heyken A, de Groot PW, Hiller E, Mock M, de Koster C, Horn U, Rupp S, Hube B (2011) Proteolytic cleavage of covalently
linked cell wall proteins by Candida albicans Sap9 and Sap10. Eukaryot Cell 10: 98-109
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Scrimale T, Didone L, de Mesy Bentley KL, Krysan DJ (2009) The unfolded protein response is induced by the cell wall integrity
mitogen-activated protein kinase signaling cascade and is required for cell wall integrity in Saccharomyces cerevisiae. Mol Biol
Cell 20: 164-175
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Seehausen O, Butlin RK, Keller I, Wagner CE, Boughman JW, Hohenlohe PA, Peichel CL, Saetre G-P, Bank C, Brannstrom A,
Brelsford A, Clarkson CS, Eroukhmanoff F, Feder JL, Fischer MC, Foote AD, Franchini P, Jiggins CD, Jones FC, Lindholm AK,
Lucek K, Maan ME, Marques DA, Martin SH, Matthews B, Meier JI, Most M, Nachman MW, Nonaka E, Rennison DJ, Schwarzer J,
Watson ET, Westram AM, Widmer A (2014) Genomics and the origin of species. Nat Rev Genet 15: 176-192
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Song X, Qiu SQ, Xu CG, Li XH, Zhang Q (2005) Genetic dissection of embryo sac fertility, pollen fertility, and their contributions to
spikelet fertility of intersubspecific hybrids in rice. Theor Appl Genet 110: 205-211
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Takahashi H, Wang S, Hayashi S, Wakasa Y, Takaiwa F (2014) Cis-element of the rice PDIL2-3 promoter is responsible for inducing
the endoplasmic reticulum stress response. J Biosci Bioeng 117: 620-623
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Tanaka K (2003) Three distinct rice cellulose synthase catalytic subunit genes required for cellulose synthesis in the secondary
wall. Plant Physiol 133: 73-83
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Thimm O, Blasing OE, Gibon Y, Nagel A, Meyer S, Kruger P, Selbig J, Muller L, Rhee SY, Stitt M (2004) Mapman: a user-driven tool
to display genomics data sets onto diagrams of metabolic pathways and other biological processes. Plant J 37: 914-939
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Tholl D (2006) Terpene synthases and the regulation, diversity and biological roles of terpene metabolism. Curr Opin Plant Biol 9:
297-304
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Thomas J, Ingerfeld M, Nair H, Chauhan SS, Collings DA (2012) Pontamine fast scarlet 4B: a new fluorescent dye for visualising
cell wall organisation in radiata pine tracheids. Wood SCI Technol 47: 59-75
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Trapnell C, Roberts A, Goff L, Pertea G, Kim D, Kelley DR, Pimentel H, Salzberg SL, Rinn JL, Pachter L (2012) Differential gene
and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat Protoc 7: 562-578
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Ünal M, Vardar F, Aytürk Ö (2013) Callose in plant sexual reproduction. In Marina Silva-Opps (Ed.), Current Progress in Biological
Research, InTech, Crotia: 319-343
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Vogel JT, Zarka DG, Van Buskirk HA, Fowler SG, Thomashow MF (2005) Roles of the CBF2 and ZAT12 transcription factors in
configuring the low temperature transcriptome of Arabidopsis. Plant J 41: 195-211
Pubmed: Author and Title
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Wakasa Y, Oono Y, Yazawa T, Hayashi S, Ozawa K, Handa H, Matsumoto T, Takaiwa F (2014) RNA sequencing-mediated
transcriptome analysis of rice plants in endoplasmic reticulum stress conditions. BMC Plant Biol 14: 101
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Wan S, Jiang L (2016) Endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in plants. Protoplasma 253: 765765
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Wang GW, He YQ, Xu CG, Zhang Q (2005) Identification and confirmation of three neutral alleles conferring wide compatibility in
inter-subspecific hybrids of rice (Oryza sativa L.) using near-isogenic lines. Theor Appl Genet 111: 702-710
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Wang J, Liu KD, Xu CG, Li XH, Zhang Q (1998) The high level of wide-compatibility of variety 'Dular' has a complex genetic basis.
Theor Appl Genet 97: 407-412
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Wang Q, Wan L, Li D, Zhu L, Qian M, Deng M (2009) Searching for bidirectional promoters in Arabidopsis thaliana. BMC
Bioinformatics 10: S29
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Weng X, Wang L, Wang J, Hu Y, Du H, Xu C, Xing Y, Li X, Xiao J, Zhang Q (2014) Grain Number, Plant Height, and Heading Date7 is
a central regulator of growth, development, and stress response. Plant Physiol 164: 735-747
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Winkelshirley B (2002) Biosynthesis of flavonoids and effect of stress. Curr Opin Plant Biol 5: 218-223
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Xiao C, Somerville C, Anderson CT (2014) POLYGALACTURONASE INVOLVED IN EXPANSION1 functions in cell elongation and
flower development in Arabidopsis. Plant Cell 26: 1018-1035
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Xu H, Xu W, Xi H, Ma W, He Z, Ma M (2013) The ER luminal binding protein (BiP) alleviates Cd2+-induced programmed cell death
through endoplasmic reticulum stress-cell death signaling pathway in tobacco cells. J Plant Physiol 170: 1434-1441
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Yamagata Y, Yamamoto E, Aya K, Win KT, Doi K, Sobrizal, Ito T, Kanamori H, Wu J, Matsumoto T, Matsuoka M, Ashikari M,
Yoshimura A (2010) Mitochondrial gene in the nuclear genome induces reproductive barrier in rice. Proc Natl Acad Sci U S A 107:
1494-1499
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Yang J, Sardar HS, Mcgovern KR, Zhang Y, Showalter AM (2007) A lysine-rich arabinogalactan protein in Arabidopsis is essential
for plant growth and development, including cell division and expansion. Plant J 49: 629-640
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Yang J, Zhao X, Cheng K, Du H, Ouyang Y, Chen J, Qiu S, Huang J, Jiang Y, Jiang L, Ding J, Wang J, Xu C, Li X, Zhang Q (2012) A
killer-protector system regulates both hybrid sterility and segregation distortion in rice. Science 337: 1336-1340
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Yu Y, Zhao Z, Shi Y, Tian H, Liu L, Bian X, Xu Y, Zheng X, Gan L, Shen Y, Wang C, Yu X, Wang C, Zhang X, Guo X, Wang J, Ikehashi
H, Jiang L, Wan J (2016) Hybrid sterility in rice (Oryza sativa L.) involves the tetratricopeptide repeat domain containing protein.
Genetics 203: 1439-1451
Pubmed: Author and Title
CrossRef: Author and Title
Downloaded
Google Scholar: Author Only Title Only Author
and Title from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
Yu Z, Kang B, He X, Lv S, Bai Y, Ding W, Chen M, Cho H-T, Wu P (2011) Root hair-specific expansins modulate root hair elongation
in rice. Plant J 66: 725-734
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Zhang GY, Feng J, Wu J, Wang XW (2010) BoPMEI1, a pollen-specific pectin methylesterase inhibitor, has an essential role in
pollen tube growth. Planta 231: 1323-1334
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Zhang J, Xu L, Wu Y, Chen X, Liu Y, Zhu S, Ding W, Wu P, Yi K (2012) OsGLU3, a putative membrane-bound endo-1,4-betaglucanase, is required for root cell elongation and division in rice (Oryza sativa L.). Mol Plant 5: 176-186
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Zhong R, Lee C, McCarthy RL, Reeves CK, Jones EG, Ye ZH (2011) Transcriptional activation of secondary wall biosynthesis by
rice and maize NAC and MYB transcription factors. Plant Cell Physiol 52: 1856-1871
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Zhou H, He S, Cao Y, Chen T, Du B, Chu C, Zhang J, Chen S (2006) OsGLU1, a putative membrane-bound endo-1,4-beta-Dglucanase from rice, affects plant internode elongation. Plant Mol Biol 60: 137-151
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.