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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.