Processes Underlying a Reproductive Barrier in indica-japonicaRice Hybrids Revealed by Transcriptome Analysis1[OPEN]

Yanfen Zhu, Yiming Yu, Ke Cheng, Yidan Ouyang, Jia Wang, Liang Gong, Qinghua Zhang, Xianghua Li,Jinghua Xiao, and Qifa Zhang2

National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan),Huazhong Agricultural University, Wuhan 430070, China

ORCID IDs: 0000-0003-1436-810 (X.L.); 0000-0003-2693-2685 (Q.Z.).

In rice (Oryza sativa), hybrids between indica and japonica subspecies are usually highly sterile, which provides a model systemfor studying postzygotic reproductive isolation. A killer-protector system, S5, composed of three adjacent genes (ORF3, ORF4,and ORF5), regulates female gamete fertility of indica-japonica hybrids. To characterize the processes underlying this system, weperformed transcriptomic analyses of pistils from rice variety Balilla (BL), Balilla with transformed ORF5+ (BL5+) producingsterile female gametes, and Balilla with transformed ORF3+ and ORF5+ (BL3+5+) producing fertile gametes. RNA sequencing oftissues collected before (MMC), during (MEI), and after (AME) meiosis of the megaspore mother cell detected 19,269 to 20,928genes as expressed. Comparison between BL5+ and BL showed that ORF5+ induced differential expression of 8,339, 6,278, and530 genes at MMC, MEI, and AME, respectively. At MMC, large-scale differential expression of cell wall-modifying genes andbiotic and abiotic response genes indicated that cell wall integrity damage induced severe biotic and abiotic stresses. Theprocesses continued to MEI and induced endoplasmic reticulum (ER) stress as indicated by differential expression of ERstress-responsive genes, leading to programmed cell death at MEI and AME, resulting in abortive female gametes. In the BL3+5+/BL comparison, 3,986, 749, and 370 genes were differentially expressed at MMC, MEI, and AME, respectively. Largenumbers of cell wall modification and biotic and abiotic response genes were also induced at MMC but largely suppressedat MEI without inducing ER stress and programed cell death , producing fertile gametes. These results have general implicationsfor the understanding of biological processes underlying reproductive barriers.

Reproductive isolation, a phenomenon widely existingin natural organisms, promotes speciation and maintainsthe integrity of species over time (Coyne and Orr, 2004).It is divided into two general categories depending onwhether it occurs before or after fertilization: prezygoticreproductive isolation and postzygotic reproductive isola-tion (Seehausen et al., 2014). The former prevents the for-mation of hybrid zygotes, while the latter results in hybridincompatibility, including hybrid necrosis, weakness, ste-rility, and lethality in F1 or later generations (Ouyang andZhang, 2013). The Dobzhansky-Muller model suggeststhat hybrid incompatibility is caused by the negative in-teractions between functionally divergent genes in theparental species (Dobzhansky, 1937; Muller, 1942).

Rice (Oryza sativa), a major food crop and modelplant for genomic study of monocotyledon species, alsoprovides a model system for studying reproductive iso-lation. The Asian cultivated rice is composed of two sub-species, indica and japonica. Their hybrids are usuallyhighly sterile,which is a barrier for utilization of the strongvigor in indica-japonica hybrids. A number of loci respon-sible for hybrid sterility have been identified, includingones that cause embryo-sac sterility, pollen sterility, andspikelet sterility (Ouyang et al., 2009). So far, six loci re-sponsible for hybrid incompatibility have been cloned.Among them, DPL1/DPL2 (Mizuta et al., 2010), S27/S28(Yamagata et al., 2010), and Sa (Long et al., 2008) are re-sponsible for pollen fertility, while hsa1 (Kubo et al., 2016),S7 (Yu et al., 2016), and S5 (Chen et al., 2008; Yang et al.,2012) control female fertility; all of them cause spikeletsterility. Three evolutionary genetic models have beenproposed to summarize the findings: parallel divergence,sequential divergence, and parallel-sequential divergence(Ouyang and Zhang, 2013). DPL1/DPL2 and S27/S28 inrice, HPA1/HPA2 in Arabidopsis (Arabidopsis thaliana),and JYAlpha in Drosophila melanogaster conform to theparallel divergencemodel. Sa, hsa1, andmany loci in otherspecies can be explained by the sequential divergencemodel.However,S5 in rice is the only case identified so farto follow the parallel-sequential divergence model.

S5 is a major locus controlling indica-japonica hybridsterility identified in many studies (Ikehashi and Araki,

1 This work was supported by grants from the National Key Re-search and Development Program (2016YFD0100903) and the Na-tional Natural Science Foundation (31130032) of China.

2 Address correspondence to qifazh@mail.hzau.edu.cn.Y.Z. and Qifa Zhang designed the research; Y.Z., Y.Y., K.C., Y.O.,

J.W., L.G., and Qinghua Zhang performed the research; X.L. and J.X.contributed reagents; Y.Z. and Qifa Zhang analyzed data and wrotethe article.

The author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Qifa Zhang (qifazh@mail.hzau.edu.cn).

[OPEN] Articles can be viewed without a subscription.www.plantphysiol.org/cgi/doi/10.1104/pp.17.00093

Plant Physiology�, July 2017, Vol. 174, pp. 1683–1696, www.plantphysiol.org � 2017 American Society of Plant Biologists. All Rights Reserved. 1683

https://plantphysiol.orgDownloaded on January 21, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.

1986; Liu et al., 1992, 1997; Wang et al., 1998, 2005; Qiuet al., 2005; Song et al., 2005; Chen et al., 2008) and is alsoassociated with the divergence and domestication of rice(Du et al., 2011; Ouyang et al., 2016). In a typical indica-japonica hybrid, the preferential abortion of female gameteswith japonica allele is regulated cooperatively by three ad-jacent genes,ORF3,ORF4, andORF5, at the S5 locus (Yanget al., 2012). Typical japonica and indica varieties carryORF3-ORF4+ORF5- and ORF3+ORF42ORF5+, respec-tively, where the functional allele of each gene is indicatedwith “+” while the nonfunctional allele as “2.” ORF5 en-codes an aspartic protease with cell wall localization. Theindica allele ORF5+ differs from the japonica allele ORF52by two nucleotides, both of which cause amino acidchanges (Chen et al., 2008). The japonica allele ORF4+ en-codes an unknown transmembrane protein, while theindica allele ORF42 has an 11-bp deletion resulting in anendoplasmic reticulum (ER) localization.ORF3 is predictedto encode a heat shock protein (HSP) resident in ER func-tioning to resolve ER stress. The japonica alleleORF32has aframeshift in the C terminus relative to the indica ORF3+due to a 13-bp deletion. In this system, extracellular ORF5+presumably catalyzes its substrate, producing a substancethat is sensed by the transmembrane protein ORF4+ thatinduces ER stress and subsequently leading to prematureprogrammed cell death (PCD). It is speculated that ORF3+,but not ORF32, can prevent/resolve the ER stress causedby ORF5+/ORF4+. Based on the results of genetic andcytological analyses, Yang et al. (2012) proposed a killer-protector model for the roles of these three genes in thissystem: ORF5+, together with ORF4+, kills the gametes byinducing ER stress that causes premature PCD. ORF3+protects the gametes frombeingkilled and thus restores thehybrids fertility by preventing/resolving ER stress.

In recent years, transcriptomic analysis based on RNA-sequencing technology has become a powerful tool tocharacterize regulatory networks and pathways to enhanceunderstanding of biological processes (Vogel et al., 2005;Digel et al., 2015;Groszmannet al., 2015;Coolen et al., 2016).In this study, we performed a comparative transcriptomicanalysis of pistils from Balilla (a typical japonica variety),BalillaORF5+ (Balilla with a transformed ORF5+), andBalillaORF3+ORF5+ (Balilla with transformed ORF3+ andORF5+)before, during, andafter themegasporemeiosis. Thecomparisons detected thousands of genes that were differ-entially regulated in these three genotypes at various stages.Functional classification of the differentially regulated genesidentified major patterns of differential expression, whichsuggested the biological processes underlying the killer-protector system of the S5 locus. These results provide in-sights into the functional rolesofORF3+,ORF4+, andORF5+genes as well as the cellular and biochemical nature of theirinteractions in regulating indica-japonica hybrid sterility.

RESULTS

Ovule Abortion Process of BL5+

Previous study (Yang et al., 2012) showed that the S5reproductive barrier is composed of three tightly linked

genes, ORF3, ORF4, and ORF5, in which ORF5+ andORF4+ together function as the killer of the female gam-etes and ORF3+ protects the gametes from killing. Atypical indica variety has the haplotype ofORF3+,ORF42,andORF5+, and japonica variety is ofORF32,ORF4+, andORF52. Balilla (abbreviated BL) is a typical japonicavariety. Transgenic plants BalillaORF5+ (abbreviatedBL5+) carrying a transformed ORF5+ under its nativepromoter showed extremely low spikelet fertility (6.50%)compared with wild-type BL (75.52%). In contrast,BalillaORF3+ORF5+ (abbreviated BL3+5+), carrying bothtransformed ORF5+ and transformedORF3+ (also underits native promoter), had normal spikelet fertility (77.35%)due to the protective role ofORF3+ (Supplemental Fig. S1,A–C). Paraffin section results showed that the ovule de-velopment process in both BL andBL3+5+was normal. Inbrief, the archesporial cell enlarged initially and devel-oped into a megaspore mother cell (Fig. 1, A and K). Themegaspore mother cell then underwent meiotic division,forming four megaspores in a line (Fig. 1, B, C, L, andM).The three megaspores near the micropyle degenerated,while the megaspore nearest the chalaza enlarged anddeveloped into a functional megaspore (FM; Fig. 1, D andN). The FM underwent three rounds of mitotic division toproduce an eight-nucleate embryo-sac (Fig. 1, E andO). Inthe aborted ovule of BL5+, the megaspore mother cell,dyad, and tetrad could be produced as in BL (Fig. 1, F–H).However, the nucellus cells showed vacuolization andeven degeneration at the meiosis stage (Fig. 1, G and H).After meiosis, morphological abnormalities of both nu-cellus cells and FMwere observed (Fig. 1I). The abnormalFM did not enlarge and failed to develop into a matureembryo-sac. The aborted embryo-sac accumulated un-known substances that were deeply stained (Fig. 1J).Thus, the megaspore meiosis process in BL5+ was nor-mal, but the nucellus cells around the megasporesshowed serious defects at the megaspore meiosis stage.The nucellus cell degradation and FM abortion causedthe sterility of BL5+.

ORF5 andORF4were neighboring genes overlappingin their promoter region (Yang et al., 2012). These kindsof gene pairs are frequently coexpressed (Wang et al.,2009; Kourmpetli et al., 2013). ORF5 and ORF4 had asimilar expression profile and relatively high expressionin reproductive organs (Yang et al., 2012). The in situhybridization showed that ORF5+ had localized ex-pression in developing ovules (Chen et al., 2008). Simi-larly,ORF4+ also displayed high and specific expressionin ovule tissues (Supplemental Fig. S2, G–K). The specificexpression of ORF5+ and ORF4+ in ovules was in ac-cordance with the specific abortion of ovules in BL5+.However, the protector ORF3+ showed a wide range ofexpression in many tissues including ovules, indicatingits possible function in many processes (SupplementalFig. S2, A–F).

Based on the ovule development process, we dividedthe ovule abortion process into three stages (Fig. 1). Thefirst was the megaspore mother cell (MMC) stage whenthe ovule had megasporocyte and no obvious abnor-malities were observed in BL5+. The second was the

1684 Plant Physiol. Vol. 174, 2017

Zhu et al.

https://plantphysiol.orgDownloaded on January 21, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.

meiosis (MEI) stage when meiosis was in progress andthe nucellus cells of BL5+ were vacuolated. The thirdwas after meiosis (AME) when some unknown sub-stances accumulated in embryo-sac (Fig. 1).

Processes Underlying Gamete Abortion in BL5+

We collected the pistils of wild-type BL, sterile BL5+,and fertility-restored BL3+5+ at MMC, MEI, and AME,and performed transcriptomic analysis. A data matrixof three stages from three genotypes was generated.A total of 19,269 to 20,928 genes were detected asexpressed in these tissues at different stages repre-senting 34.53 to 37.50% of total annotated genes of theNipponbare reference genome (Supplemental TableS1; Kawahara et al., 2013). A total of 8,339, 6,278, and530 genes were differentially expressed in BL5+compared with BL at MMC, MEI, and AME, respec-tively, including 4,234, 2,430, and 410 genes that wereup-regulated at the three stages and 4,105, 3,848, and120 genes that were down-regulated (SupplementalTable S2; Supplemental Data Set 1). Among thedifferentially expressed genes (DEGs), 2,067 wereup-regulated and 3,325 were down-regulated at bothMMC and MEI (Fig. 2A). These 5,392 common DEGsaccounted for 64.66 and 85.89% of total DEGs atMMC and MEI, respectively, indicating that MMCand MEI involved substantially overlapping cellularand molecular responses.

An Overview of the Differentially Induced Processes in BL5+Revealed by the Transcriptomes

We performed MapMan and Gene Ontology (GO)enrichment analyses to classify the DEGs. MapMan isan effective tool to analyze metabolic pathways andother biological processes of the transcriptome sys-tematically (Thimm et al., 2004; Ramsak et al., 2014). Toget a global view of the 8,339 DEGs in BL5+ at MMC,we used the “Metabolism overview” and “Cellularresponse” of MapMan to outline the transcriptionalchanges of genes with putative functions in metabo-lism and cellular response (Fig. 2, B and C).

In “Metabolism overview,” primary metabolic pro-cesses, including cell wall (354 genes), amino acid(226 genes), lipids (316 genes), nucleotide (81 genes), andmajor carbohydrate (128 genes) metabolism, accountedfor 13.25% of total DEGs. The cell wall localization ofORF5+, whichwas previously hypothesized to initiate thepremature PCD (Yang et al., 2012), led us to focus ourattention on the cell wall metabolic genes. We found that187 (128 up-regulated and 59 down-regulated) of the354 genes were involved in cell wall degradation andmodification, which may affect the cell wall structuresin one way or another. The secondary metabolismprocesses, including terpenes (64 genes), flavonoids(105 genes), and phenylpropanoids (120 genes) me-tabolism, which are important in plant stress response(Dixon and Paiva, 1995; Winkel-Shirley, 2002; Tholl,

Figure 1. The ovule development process of BL, BL5+, and BL3+5+. A to E, Ovule development of BL (gametes fertile).F to J, Ovule development of BL5+ (gametes sterile). K to O, Ovule development of BL3+5+ (gametes fertile). A, F, K, TheMMC stage. B, G, L, The dyad stage. C, H, M, The tetrad stage. D, I, N, The FM stage. E, J, O, The mature embryo sac. Dy,dyad; Te, tetrad; DM, degenerated megaspore; AFM, aborted functional megaspore; ANC, abnormal nucellus cells; ES,embryo sac; AES, aborted embryo sac. Bars = 30 mm. The gray bars at the top indicate the corresponding stage intranscriptome data.

Plant Physiol. Vol. 174, 2017 1685

Transcriptomic Basis of a Rice Reproductive Barrier

https://plantphysiol.orgDownloaded on January 21, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.

2006), constituted 3.47% of all DEGs. In the term of“Cellular response,” 8.65% (721 genes) of DEGs wereresponsive to biotic and abiotic stress; 1.94% (162 genes)of DEGs were related to redox, a phenomenon usuallytriggered by stress; and 6.67% (667 genes) of DEGsfunctioned in developmental processes.

GO analysis for up-regulated genes revealed that threeGO terms related to cell wall metabolism, seven GOterms involved in stress or stimulus response, and elevenGO terms associated with gene expression, protein syn-thesis, and translation were significantly enriched (falsediscovery rate [FDR] , 0.005; Fig. 3).

The change in cell wall structure by cell wall modifi-cation genes may cause cell wall integrity damage, which

was similar to the situation of pathogen or pest attack.Wethus explored the “Biotic stress” in BL5+ at MMC andfound that DEGs in various functional categories of bioticstress were enriched, including R genes, pathogen-relatedproteins, signaling, hormone signaling, proteolysis, ROSregulation, and biotic stress-responsive transcription fac-tors (Supplemental Fig. S3A).

Thus, both theMapMan andGO results indicated thata severe stress response including both biotic and abioticstresses was induced in BL5+ at MMC, which may becaused by the damage in cell wall integrity, even thoughno abnormalities were observed at this stage (Fig. 1F).

At MEI, 163 of the 300 DEGs in the “cell wall” wererelated to the cell wall degradation and modification

Figure 2. Analysis of genes differentially expressed in BL5+ compared to BL. A, Numbers of the up- and down-regulated DEGs atMMC andMEI. B, Numbers of DEGs in different MapMan functional categories at MMC,MEI, and AME, respectively. C, The heatmap of “Metabolism overview” and “Cellular response” in BL5+ at MMC based on the MapMan results. Each small squarerepresents a gene differentially expressed in BL5+ versus BL. The color of the square represents the level of up-regulation (red) ordown-regulation (green) based on the log2 fold change of the DEGs.

1686 Plant Physiol. Vol. 174, 2017

Zhu et al.

https://plantphysiol.orgDownloaded on January 21, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.

including 102 up-regulated and 61 down-regulated DEGs(Fig. 2, B and C). Cell wall-related GO terms such as“glucan metabolic process,” “polysaccharide metabolicprocess,” and “polysaccharide biosynthetic process”wereenriched (Fig. 3). The continuous up-regulation of cellwall-degrading and -modifying genes at MMC and MEImight be related to the observed cell wall abnormalitiessuch as vacuolization and degradation at MEI (Fig. 1, Gand H). In addition, 599 DEGs (9.54% of total DEGs) in“biotic stress” and “abiotic stress” and genes in various

functional categories of biotic stress were identified (Fig.2B; Supplemental Fig. S3B), suggesting that the stress re-sponse also continued at MEI.

By the AME stage, DEG numbers in all the metabolicprocesses and cellular responses were largely reducedcompared to those at MMC and MEI (Fig. 2B). The ovulehad aborted with the accumulation of unknown sub-stances. In contrast, however, a GO term “cellulose bio-synthetic process” was significantly enriched (Fig. 3),indicatingmisregulation of cellulose biosynthesis in BL5+.

Large-Scale Transcriptional Changes of Cell Wall-Modifyingand -Degrading Genes in BL5+ at MMC and MEI

The MapMan and GO results showed that cell wallmetabolism, especially cell wall modification and degra-dation, were active in BL5+ (Fig. 2). Cell wall-modifyingand -degrading genes, also called cell wall-remodelinggenes, affect cell growth by changing the extensibilityand physiochemical properties of the cell wall (Cosgrove,1999; ZhiMing et al., 2011; Gou et al., 2012; Hepler et al.,2013; Xiao et al., 2014). We analyzed the expression ofgenes from seven main enriched families: expansins(EXPs), xyloglucan endotransglucosylase/hydrolases(XTHs), pectin methyl esterases (PMEs), pectin acety-lesterases (PAEs), polygalacturonases (PGases), gly-coside hydrolase family 9 (GH9), and arabinogalactanproteins (AGPs), which affect cell wall structure andextension by enzymatic or nonenzymatic ways (Boschet al., 2005; Coimbra et al., 2008; Maris et al., 2009; Gouet al., 2012; Che et al., 2016). We found that 95, 85, and23 genes from these families were differentially expressedat MMC, MEI, and AME, respectively, of which 73 geneswere differentially expressed at both MMC and MEI, in-cluding 59 up-regulated and 14 down-regulated (Fig. 4, Aand B). The 59 up-regulated genes consisted of 13 EXPs,7 XTHs, 5 PMEs, 4 PAEs, 8 PGases, 7 GH9s, and 15AGPs.OsEXP4was elevated in BL5+ at MMC and MEI. In rice,overexpression ofOsEXP4 results in increased cell length,while repression of OsEXP4 reduces cell length (Choiet al., 2003). Three up-regulated XTHs (Os06g48160,Os03g01800, andOs10g39840) have been characterized toutilize xyloglucan, one of the cell wall components, assubstrate (Hara et al., 2014).OsGLU1, a GH9member thatregulates cell elongation in rice (Zhou et al., 2006;Zhang et al., 2012), was found up-regulated as well.The up-regulation of genes involved in cell wall degra-dation and cell size regulationwas in accordancewith cellwall abnormalities in BL5+ (Fig. 1, G and H). Thus, weinferred that the up-regulation of the cell wall-remodelinggenes induced by ORF5+ in BL5+ led to cell structurechange, damaging the cell wall integrity.

Cellulose Deposition in BL5+

In BL5+, unknown substances were accumulated inthe aborted ovule at AME (Fig. 1, I and J). The enrich-ment of the GO term “cellulose biosynthetic process”suggested a misregulation of cellulose biosynthesis andthat the deposited substances in the aborted ovule

Figure 3. Partial significantly (FDR , 0.005) enriched GO terms forup-regulated genes in BL5+ compared to BL. The color in each cellrepresents the significance of enrichment based on the FDR value. Thecells without color indicate not significantly enriched. The full list ofGO terms is given in Supplemental Table S3.

Plant Physiol. Vol. 174, 2017 1687

Transcriptomic Basis of a Rice Reproductive Barrier

https://plantphysiol.orgDownloaded on January 21, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.

might overaccumulate cellulose. To validate this, we usedPontamine fast scarlet 4B and calcofluor white to staincellulose (Anderson et al., 2010; Thomas et al., 2012), andobserved strong fluorescence signals in BL5+ at the sitewhere the substances accumulated, indicating that cellu-lose was indeed in large quantity (Fig. 5, B and E), whichwas also supported by the much higher cellulose contentin panicles of BL5+ than in BL (Fig. 5G). In contrast, nor-mal mature embryo-sac structures were easily identifiedin the ovule of BL and BL3+5+, where low fluorescenceintensity was detected (Fig. 5A, C, D, and F).

Cellulose is amajor cellwall polysaccharide composed ofunbranched b-1, 4-linked glucan chains. In rice, OsMYB46and OsSWNs are secondary cell wall biosynthetic regula-tors.OsSWN1acts on theupstreamofOsMYB46bybindingto the SNBEelement in its promoter region.Overexpressionof OsSWN1 or OsMYB46 in Arabidopsis results in theectopic deposition of cellulose, xylan, and lignin (Zhonget al., 2011). In BL5+, OsSWN1, OsSWN4, OsSWN6, andOsMYB46 were up-regulated at MMC and MEI. Three

cellulose synthases, OsCESA4, OsCESA7, and OsCESA9,which are regarded as subunits of cellulose synthase com-plex (Tanaka et al., 2003), were continuously up-regulatedfromMMC toAME. In addition, expression offive cellulosesynthase-like genes (OsCslA1, OsCslA3, OsCslA5, OsCslA9,andOsCslF6) and two brittle culmgenes (BC1 andBC3)werealso elevated at MMC or MEI (Fig. 4C). Upstream tran-scription factors such asOsSWN1,OsSWN6, andOsMYB46had peak expression atMEI, while their downstream genessuch asOsCESAs and two brittle culm genes had the highestexpression at AME (Supplemental Fig. S4), in agreementwith theoveraccumulationof cellulose atAME(Fig. 5, BandE). These results showed that the cellulose biosyntheticpathway was highly activated in BL5+, resulting in the ac-cumulation of cellulose in the aborted embryo-sac.

Callose Deposition in BL5+

Callose is a dynamic component of the plant cell wallthat plays important roles in megasporogenesis. The

Figure 4. Differential regulation of cell wall genes in BL5+ and BL3+5+ compared with BL. A, The number of differentiallyexpressed genes involved in cell wall modification and degradation in BL5+ and BL3+5+. The x axis indicates the gene numbers.The y axis indicates the gene families at different stages. B, Heat map of expression change of genes involved in cell wallmodification and degradation. The color in each cell represents the level of expression change based on the log2 fold. The cellwithout color indicates the gene was not differentially expressed. Full list of genes and their expressions is given in SupplementalTable S5. C, Expression change of cellulose biosynthetic genes.

1688 Plant Physiol. Vol. 174, 2017

Zhu et al.

https://plantphysiol.orgDownloaded on January 21, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.

uneven distribution of the callose wall around the meg-aspores decides the polarization of megaspores, the for-mation of a functional megaspore, and the abortion of theother three nonfunctional megaspores (Ünal et al., 2013).Since many genes encoding b-1,3-glucanases, which haveputative functions in callose metabolism, were differen-tially expressed in BL5+ at MMC andMEI (SupplementalFig. S5), we investigated the dynamic callose distributionsin ovule of BL, BL5+, and BL3+5+. Duringmeiosis, callosepreferentially accumulated at the micropylar end ofmegaspores. Two callose bandswere observed in all threegenotypes at the dyad stage (Fig. 6, A–F), and at the tetradstage at least three callose bands could be recognized alsoin all three genotypes although slightly different in dis-tribution patterns (Fig. 6, G–L). This apparent normalcallose distribution inmegaspores of BL5+ indicated likelynormal meiosis. Unlike BL and BL3+5+, however, weakcallose deposition was detected in nucellus cells at thetetrad stage in BL5+ (Fig. 6I). At the functional megaspore(FM) stage, callose accumulated in the three degenerated

gametes but not in the FM (Fig. 6, M–R). The survived FMsubsequently developed to embryo-sac (Fig. 6, S, T, W,and X). However, the ovule of BL5+ displayed irregularcallose deposition around the FM and in nucellus cells(Fig. 6, O and P), and finally, the aborted embryo-sac wasstuffed with a large amount of callose (Fig. 6, U and V).Thus, ORF5+ resulted in a misregulation of callose depo-sition duringmegasporogenesis such that callose began toaccumulate abnormally fromMEI and was deposited in alarge quantity at AME in the ovule of BL5+.

ER Stress and Premature PCD Induced in BL5+

Under stress conditions, the number of misfoldedproteins will increase and may exceed the capacity ofthe ER quality control system, thus inducing ER stress(Howell, 2013; Wan and Jiang, 2016). In BL5+, 78.95%of genes in “RNA transcription,” 66.23% of genes in“RNA processing,” 57.69% of genes in “protein syn-thesis initiation,” 58.33% of genes in “protein synthesis

Figure 5. Cellulose deposition in themature ovulesof BL, BL5+, and BL3+5+. A to F, Cellulose depo-sition in BL (A and D), BL5+ (B and E), and BL3+5+(C and F). A to C, Pontamine fast scarlet 4B staining.D to F, Calcofluor white staining. The strong red orblue fluorescence indicates where the celluloseaccumulated. Bars = 50mm.G, Cellulose content inthe panicles of BL and BL5+. The data aremeans 6 SE (n = 3). ***Significant difference atP , 0.001.

Figure 6. Callose deposition (green fluores-cence) in the developing ovule of BL, BL5+,and BL3+5+ by aniline blue staining. A to F,The dyad stage. The red arrowheads indicatethe callose bands around the dyad. G to L,The tetrad stage. The red arrowheads indicatethe callose bands around the tetrad. M to R,The functional megaspore stage. The red ar-rowheads indicate the degenerated mega-spores. S to X, The mature embryo-sac. I, O,and U, The gray arrowheads indicate abnor-mally deposited callose. Bars = 25 mm.

Plant Physiol. Vol. 174, 2017 1689

Transcriptomic Basis of a Rice Reproductive Barrier

https://plantphysiol.orgDownloaded on January 21, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.

elongation,” 82.46% of genes in “ribosomal protein syn-thesis,” and 60.91% of genes in “ribosome biogenesis”were up-regulated at MMC (Supplemental Fig. S6), sug-gesting a greatly increased level of protein synthesis,which may increase the protein folding burden on ER. Todetect whether ER stress occurred in BL5+ at MMC, weexamined the expression of ER stress-responsive genes inBL5+ and found that 10 such genes were significantlyup-regulated, including OsbZIP50, which is a key ERstress regulator in rice (Fig. 7A). The mRNA of OsbZIP50was splicedwhenER stress occurred (Hayashi et al., 2012).We thendetected the unconventional splicing ofOsbZIP50mRNA in different materials and found that OsbZIP50mRNA was spliced in BL5+ at MMC, but not in BL orBL3+5+ (Fig. 7B). The splicedOsbZIP50mRNA encodes atranscription factor OsbZIP50-S, which binds to thepUPRE-II cis-element in the promoter regions of OsBIP4,ORF3, OsEBS, and OsPDIL2-3 (Hayashi et al., 2013;Takahashi et al., 2014; Wakasa et al., 2014). Accordingly,expression of these four genes was elevated (Fig. 7A).Some other genes including Fes1-like, CRT2-2, CRT1, andSAR1B-like, which were induced in ER stress, were also

up-regulated. These results showed that ER stress oc-curred in BL5+ at MMC relative to BL and BL3+5+. Inresponse to ER stress, genes involved in protein modifi-cation (303 up-regulated and 358 down-regulated) andtargeting (70 up-regulated and 53 down-regulated) weredifferentially expressed, presumably to help protein pro-cessing and transportation. In addition, 793DEGs (339 up-regulated and 454 down-regulated) involved in “proteindegradation” were found, likely as a remedy to degradethe misfolded or unfolded proteins (Supplemental Fig.S6). The splicing ofOsbZIP50mRNAandup-regulation ofER stress-responsive genes were also detected at MEI andAME (Fig. 7, A andB), indicating that ER stress proceededcontinuously in BL5+.

The unresolved ER stress could induce premature pro-grammed cell death (PCD) (Cai et al., 2014). In BL5+, PCDsignals were detected from the meiosis stage (Yang et al.,2012), corresponding to the MEI stage in this study. AtMEI,wedetected up-regulation of two aspartic protease-encoding genes, OsAP25 and OsAP37 (Fig. 7, A and C),both of which participate in the tapetal PCD processand induce PCD in yeast and plants (Niu et al., 2013).

Figure 7. Differential regulation of ER stress and PCD genes. A, Log2 fold change of the expression level of ER stress and PCDgenes in BL5+ against BL. The color in each cell represents the levels of expression change based on the log2 fold. The cell withoutcolor indicates the gene was not differentially expressed. B, Splicing ofOsbZIP50mRNA in BL, BL5+, and BL3+5+ at each stage.C, qPCR verification for expression of tapetal PCD-related genes in BL and BL5+. The x axis represents different stages. The y axisrepresents the expression level relative to profilin (Os06g05880). The data are means 6 SE (n = 3).

1690 Plant Physiol. Vol. 174, 2017

Zhu et al.

https://plantphysiol.orgDownloaded on January 21, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.

Overexpression ofOsAP25 orOsAP37 in plants results incell death, which is suppressed by aspartic protease-specific inhibitor pepstatin A. Transformation of OsAP25or OsAP37 into a PCD-deficient yeast strain restores thePCD ability of the strain. OsAP25 showed the highestexpression at MEI (Fig. 7C), consistent with the observa-tion of PCD signals at this stage. In the tapetal PCDprocess, transcription factor EAT1 directly regulatesthe expression of OsAP25 and OsAP37 (Niu et al.,2013). However, EAT1 was down-regulated at MEI,indicating that some other regulatorsmight have replacedEAT1 to activate OsAP25 and OsAP37. TDR and TIP2,which work upstream of OsAP25 and OsAP37 in pollendevelopment (Niu et al., 2013), were up-regulated in BL5+at MMC and MEI (Fig. 7, A and D). The coordinatedup-regulation of TDR, TIP2, OsAP25, and OsAP37suggested that the ovule and tapetummay share somecommon genes/pathways in PCD. Taken together, an

OsbZIP50-dependent ER stress pathway and a pre-mature PCD pathway involving the tapetal PCD geneswere activated in BL5+.

These results suggested that the ORF5+-inducedup-regulation of cell wall degradation and modifica-tion genes in BL5+ caused cell wall damage, whichinduced both biotic and abiotic stresses. Lasting stressresponses induced ER stress and unresolved ER stressled to premature PCD, eventually resulting in the ovuleabortion.

Processes Detected in the BL3+5+/BL Comparison

BL3+5+ carrying both the killer ORF5+ and the pro-tectorORF3+ showed no phenotypic difference from thewild-type BL with respect to fertility (Supplemental Fig.S1). However, 3,986 DEGs were still identified between

Figure 8. Analysis of genes differentially expressed in BL3+5+ compared to BL. A, Number of DEGs detected in the BL3+5+/BLand BL5+/BL comparisons at different stages. The overlapping areas indicate the genes differentially expressed in both BL3+5+and BL5+. B, The number of DEGs in different functional categories of “Metabolism overview” and “Cellular response” byMapMan atMMC,MEI, andAME, respectively. C, Partial significantly (FDR, 0.005) enrichedGO terms for up-regulated genes inBL3+5+ compared with BL. The color in each cell represents the significance of enrichment based on the FDR value. The cellswithout color indicate not significantly enriched. The full list of GO terms is given in Supplemental Table S4.

Plant Physiol. Vol. 174, 2017 1691

Transcriptomic Basis of a Rice Reproductive Barrier

https://plantphysiol.orgDownloaded on January 21, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.

BL3+5+ and BL at MMC (Supplemental Table S2;SupplementalData Set 2), ofwhich 3,843DEGswere sharedwith the BL5+/BL comparison at this stage, indicatingthat ORF5+ induced similar processes in BL3+5+ (Fig.8A). As expected, the process of “cell wall”was enrichedwith 217 DEGs (Fig. 8B), of which 64 (55 up-regulatedand 9 down-regulated) were cell wall-modifyingand -degrading genes from the seven main families(Fig. 4, A and B). GO terms “glucanmetabolic process”and “polysaccharide metabolic process” were signifi-cantly enriched in up-regulated genes (Fig. 8C). Terms“biotic stress” and “abiotic stress” were also enrichedinvolving 339 DEGs. However, the number of DEGs ineach of the enriched functional categories was smallerthan that in the BL5+/BL comparison (Fig. 4, A and B).Moreover, the splicing of OsbZIP50 mRNA and theup-regulation of its downstream genes were not detected(Fig. 7, A and B). These results suggested that ORF5+induced similar processes in BL3+5+ at MMC, especiallywith respect to cell wall integrity damage, but to a muchlesser extent, which did not trigger ER stress.

At MEI, the difference between BL3+5+ and BL wasgreatly narrowed such that only 749 DEGs were identi-fied, compared with 6,278 genes in the BL5+/BL com-parison (Supplemental Table S2; Supplemental Data Set2). Thus, numbers of DEGs, especially those in cell wall,biotic stress, and abiotic stress, were greatly reduced (Fig.8B). Only seven DEGs from the seven cell wall familieswere found (Fig. 4, A and B), suggesting that cell wall

integrity damage was suppressed. The cell wall com-pensation pathways such as cellulose biosynthesis andcallose deposition were not activated (Figs. 4C and 6, Kand Q), and ER stress and PCD did not occur (Fig. 7). Asa result, the cell wall maintained integrity and the ovulemeiosis was undisturbed (Fig. 1, L and M).

At AME, the number of DEGs was further reduced to370 in BL3+5+ compared with BL (Supplemental TableS2; Supplemental Data Set 2). These results indicatedthat ORF5+ induced similar but weaker damage pro-cesses in BL3+5+ as in BL5+ at MMC, but these pro-cesses were soon suppressed by ORF3+. Consequently,the embryo-sac developed normally, producing fertilefemale gametes (Fig. 1O).

DISCUSSION

The killer-protector system of S5 is composed of threeproteins: cell wall protein ORF5+, transmembrane proteinORF4+, and ER resident protein ORF3+. Based on thecomparative transcriptome results of BL5+ and BL3+5+,we proposed a model to summarize the processes under-lying the S5 system (Fig. 9).

In BL5+, many cell wall-modifying and -degradinggenes in the families EXPs, XTHs, PMEs, PAEs, PGases,GH9, and AGPs were up-regulated at MMC and MEI,and many genes of these families play important rolesin cell elongation or cell expansion (Zhou et al., 2006;Chen and Ye, 2007; Yang et al., 2007; Coimbra et al., 2008;Maris et al., 2009; Zhang et al., 2010, 2012; ZhiMing et al.,2011; Gou et al., 2012; Ma et al., 2013; Miedes et al., 2013;Hara et al., 2014; Xiao et al., 2014; Che et al., 2016). It hasbeen suggested that postsyntheticmodifications of the cellwall by cell wall-modifying genes may affect cell wallintegrity (CWI; Pogorelko et al., 2013). Thus, large-scaleup-regulation of cell wall-remodeling genes induced byORF5+may lead to changes of cell wall structures, whichaffect CWI, thus inducing stress response.

ORF5+ encodes an extracellular aspartic protease. InPichia pastoris and Saccharomyces cerevisiae, extracellularaspartic protease yapsins are required for the CWImaintenance (Krysan et al., 2005; Guan et al., 2012).Two plant aspartic proteases, A36 and A39, participatein cell wall remodeling in pollen tube (Gao et al., 2017).The study in Candida albicans shows that secretedaspartic proteinases use a set of cell wall proteins assubstrates (Schild et al., 2011). We speculated that oneormore substrates of ORF5+ existed in the rice cell wall,which induced cell wall damage after activation byORF5+. In Saccharomyces cerevisiae, CWI damage trig-gers unfolded protein response, a homeostatic responseto alleviate ER stress, which in turn affects the CWI(Krysan, 2009; Scrimale et al., 2009). In plants, pathogeninvasion or pest attack can induce CWI damage andsubsequent stress response (Bellincampi et al., 2014;Pogorelko et al., 2013). CWI damage eventually leads toPCD (Paparella et al., 2015). In BL5+, the functionalcategories responsive to pathogen invasion or pest at-tack were enriched, and ER stress was accordingly

Figure 9. A model for S5 locus-mediated ovule abortion.

1692 Plant Physiol. Vol. 174, 2017

Zhu et al.

https://plantphysiol.orgDownloaded on January 21, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.

induced, presumably by CWI impairment, which inturn exacerbated the cell wall damage and its subse-quent stress responses, thus finally leading to PCD. Toalleviate the adverse effects caused by CWI damage,cells usually trigger a cell wall compensatory pathwayto strengthen the cell wall (Hamann, 2012). For exam-ple, disruption by aspartic protease Yps7p in Pichiapastoris reduces chitin content in the lateral cell wall, butthe cell thickens the inner cell wall for compensation(Guan et al., 2012). Similarly, in BL5+, key genes insecondary cell wall biosynthesis were significantlyup-regulated, resulting in the cellulose accumulation torepair cell wall. Callose, a polysaccharide usually ac-cumulated under stress conditions to provide physicalbarrier to the adverse environments (Pirselova andMatusikova, 2012), was deposited in large quantity.Despite other roles of callose in ovule development, thelarge amount deposition of callose in BL5+ was mostlikely to strengthen cell wall.ORF4+ acted as a partner of ORF5+ with a transmem-

brane localization.We speculated thatORF4+ functionedas a sensor of CWI damage. In plants, a well-studied cellwall sensor is AtWAK1, a wall-associated kinase, whichresides in the plasma membrane and responds to oligo-galacturonides derived from the hydrolysis of cell wallcomponent (Brutus et al., 2010). Xa4, a wall-associatedkinase in rice, contributes to disease resistance by strength-ening the cell wall via promoting cellulose synthesis andsuppressing cell wall loosening (Hu et al., 2017). Unlikewall-associated kinase, no extracellular binding domain orinner kinase domain was found in ORF4+; thus there mayexist a coreceptor that interacts with ORF4+ in the plasmamembrane and/or a downstream signal protein that inter-acts with plasma membrane receptor to transmit signals.Based on these analyses, it was deduced that ORF5+

damaged CWI by cleaving its substrates, leading toup-regulation of the cell wall-modifying and -degradinggenes. ORF4+ sensed the CWI impairment and inducedbiotic and abiotic stress responses, and consequently in-duced ER stress due to the accumulation of drasticallyincreased misfolded proteins in ER. The occurrence of ERstress in turn deteriorated the CWI damage and led to amore serious stress response. Finally, the unresolved cellwall damage, stress response, and ER stress triggeredPCD.Meanwhile, the cellulose and callose accumulated inthe ovule as the cells’ unsuccessful remedy to repair thedamaged cell wall.BL3+5+ showed no difference with BL with respect

to ovule development and fertility, but a number ofcell wall-modifying and -degrading genes were stillup-regulated in BL3+5+ at MMC, which may induceCWI damage. However, transcriptional change ofthese genes was suppressed at MEI and AME. Thus,the subsequent negative effects caused by CWI dam-age, including ER stress, PCD, cellulose, and callosedeposition, were prevented in BL3+5+. ORF3+ en-codes a HSP 70 chaperone, which is a downstreamtarget of OsbZIP50s and induced by ER stress. ORF3+is believed to help protein folding in ER to resolve ERstress like OsBIP1-OsBIP5 in the HSP70 gene family

(Yang et al., 2012; Hayashi et al., 2013; Wakasa et al.,2014). BIP protein alleviates Cd2+ stress-induced PCD(Xu et al., 2013). The tobacco (Nicotiana tabacum) plantswith overexpression of BIP show delayed leaf senes-cence (Carvalho et al., 2014). In BL3+5+, ORF3+ sup-pressed the ORF5+-induced ER stress and PCD. It wasspeculated that one role of ORF3+ was alleviating ERstress thus not to induce premature PCD. The chape-rone activity of ORF3+may also help the folding of thesignal proteins downstream of ORF5+ and ORF4+ sothat the stress response may be suppressed at MEI.

In conclusion, we revealed the processes underlying areproductive barrier composed of ORF3, ORF4, andORF5 at the transcriptomic level and proposed a mecha-nistic model based on the results. Nevertheless, furtherstudies are needed to support and enrich the model.

MATERIALS AND METHODS

Sample Preparation and Library Construction

Balilla was a typical japonica rice (Oryza sativa) variety. The transgenic plantBalillaORF5+wasgeneratedby introducingORF5+ froman indicavarietyNanjing11 into Balilla under its native promoter (Chen et al., 2008). The plant BalillaORF3+ORF5+ was isolated from the hybrid offspring between BalillaORF5+ andBalillaORF3+, which contained a transformed ORF3+ from Nanjing 11 under itsnative promoter (Yang et al., 2012). All the rice plants were grown in the fields ofthe experimental stationofHuazhongAgriculturalUniversity,Wuhan,China.Wedetermined the relationship between the floret length and stage of ovule devel-opment by the paraffin section observation of florets from the variety Balilla. Thepistils from florets of Balilla, BalillaORF5+, and BalillaORF3+ORF5+ ranging inlength from 3.8 to 4.2 mm, 5.5 to 6.0 mm, and 7.0 to 7.5 mm, respectively, werecollected as MMC, MEI, and AME stage samples with two biological replicates.RNA was extracted using TRIzol reagent (Invitrogen) according to the manu-facturer’s manual. RNA yields and quality were measured using Nanodrop2000 (Thermo Scientific) and further quantified using Qubit2.0 Fluorometer(Invitrogen). RNA integrity was evaluated using Experion RNA Analysis Kits(Bio-Rad) and Experion Automated Electrophoresis Station (Bio-Rad). Each li-brary was constructed using 3 mg total RNA using the TruSeq RNA LibraryPreparation Kit v2 (Illumina). The libraries were sequenced using IlluminaHiSEquation 2000 sequencing platform at National Key Laboratory for CropGenetic Improvement in Huazhong Agricultural University, Wuhan, China.

Data Processing and DEG Identification

The data of the two replicates of all samples were collected and analyzedfollowing the workflow described previously (Trapnell et al., 2012). In brief,the raw sequencing reads were mapped to the rice reference genome(MSU7.0, http://rice.plantbiology.msu.edu/index.shtml) using TopHat,then the transcripts were assembled and quantified using Cufflinks, and fi-nally, differentially expressed genes were analyzed using Cuffdiff. The rawdata files can be found in the NCBI database under the accession numberGSE95200. Genes with$2-fold expression up-regulation or down-regulationand adjusted P value # 0.05 (counted using Cuffdiff) were regarded asup-regulated or down-regulated genes.

GO and MapMan Analysis

GO enrichment was performed in AgriGO, a web-based tool for GO analysis(Du et al., 2010; http://bioinfo.cau.edu.cn/agriGO/index.php). The species “Oryzasativa”was chosen. GO terms in biological process groups with FDR, 0.005 wereidentified and analyzed further. The MapMan software version 3.5.1.R2 andpathways were downloaded from the MapMen Site of analysis (http://mapman.gabipd.org/web/guest/mapmanstore). The mapping information of rice genes(osa_MSU_v7_2015-09-08_mapping.txt.tar.gz) that can be imported to theMapMan softwarewas downloaded fromGoMapMan (http://www.gomapman.org/export/current/mapman; Ramsak et al., 2014).

Plant Physiol. Vol. 174, 2017 1693

Transcriptomic Basis of a Rice Reproductive Barrier

https://plantphysiol.orgDownloaded on January 21, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.

In Situ Hybridization

The methods for preparation of paraffin embedded sections followed thepreviously described protocol (Weng et al., 2014). Primer pairs (listed inSupplemental Table S4) ORF3insituF and ORF3insituR, ORF4insituF andORF4insituR were used to prepare probes for ORF3 and ORF4, respectively.Products amplified from ORF3+ or ORF4+ cDNA were ligated to pGEM-Tvector (Promega). The sense probe was transcribed with T7 RNA polymerase,and the antisense probe was transcribedwith SP6 RNA polymerase. The probeswere labeled with digoxigenin (Roche). Hybridization and detection wereperformed as described previously (De Block and Debrouwer, 1993).

PCR and RT-qPCR

Total RNA of pistils was isolatedwith TRIzol reagent (Invitrogen) and treatedwithRNase-freeDNaseI (Invitrogen) for 20min at room temperature todigest thecontaminating DNA. The first-strand cDNAwas synthesized using SSIII reversetranscriptase (Invitrogen) with Oligo(dT)15 Primer (Promega) following themanufacturers’ instructions. For amplification of OsbZIP50s, specific primersaround the splice site were used. PCR conditions and PCR product detectionwere performed as described before (Yang et al., 2012). For quantitative real-timePCR, we carried out the reaction in a total volume of 10 mL containing 5 mLFastStart Universal SYBR Green Master (Rox; Roche), 1 mL cDNA sample, and3 mM each primer on ABI ViiA7 system. The primers for PCR and RT-qPCR arelisted in Supplemental Table S4. The gene profilin (Os06g05880) was used as theinternal control in RT-qPCR (Yang et al., 2012).

Histological Staining

The freshfloretswerefixedwith FAA solution (50% ethanol, 5% acetic acid, and3.7% formaldehyde) at 4°C overnight, dehydrated with ethanol in a series of con-centrations (50, 70, 85, 95, and 100%), infiltratedwith xylene, and finally embeddedin paraffin. The materials were cut into 10-mm-thick sections and then washed in axylene/ethanol series (0–50% ethanol). For callose staining, the rehydrated sectionswere stained with 0.1% aniline blue solution for 60 min. For cellulose staining, therehydrated sectionswere stainedwith 0.01% Pontamine Fast Scarlet 4B solution for20 min or 0.1% calcofluor white (Sigma-Aldrich) for 5 min. The stained sectionswere scanned under fluorescence microscopy. The florets for observation of ovuledevelopment process were stained with ehrlich hematoxylin. After fixation, thematerials were rehydrated, infiltrated with ehrlich hematoxylin for 4 d, thendehydrated with ethanol, infiltrated with xylene, and embedded in paraffin. The10-mm-thick sections were dewaxed with xylene and sealed with Canada balsam(Sigma-Aldrich). The slides were scanned under light microscopy.

Cellulose Content Determination

The cellulose contentwas determined as described previously (Guo et al., 2014).In brief, the materials were suspended with potassium phosphate buffer (pH 7.0),chloroform-methanol (1:1, v/v), dimethylsulphoxide-water (9:1, v/v), 0.5% (w/v)ammonium oxalate, and 4 M KOH containing 1.0 mg/mL sodium borohydridein turn. The remaining pellet was collected and extracted further with H2SO4(67%, v/v), and the supernatants were collected for the determination of cellulose.

Accession Numbers

Sequence data from this article can be found in the GenBank libraries underaccession numbers JX138499.1 (ORF31), JX138498.1 (ORF32), JX138502.1 (ORF41),JX138503.1 (ORF42), EU889295 (ORF51), and EU889294.1 (ORF52).

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. The fertility of BL, BL5+, and BL3+5+.

Supplemental Figure S2. RNA in situ hybridization of ORF3+ and ORF4+.

Supplemental Figure S3. DEGs in “Biotic stress” in BL5+ compared withBL at MMC and MEI.

Supplemental Figure S4. qPCR verification for expression of the cellulosebiosynthetic genes in pistils of BL, BL5+, and BL3+5+ at MMC, MEI, andAME, respectively.

Supplemental Figure S5. Expression change of genes involved in callosemetabolism.

Supplemental Figure S6. The number of DEGs involved in RNA transcrip-tion, protein synthesis, and processing.

Supplemental Table S1. The number of expressed genes in BL, BL5+, andBL3+5+ at different stages.

Supplemental Table S2. The number of differentially expressed genesidentified in different comparisons.

Supplemental Table S3. Full list of significantly enriched GO terms(FDR , 0.005) for DEGs in BL5+ versus BL.

Supplemental Table S4. Full list of significantly enriched GO terms(FDR , 0.005) for DEGs in BL3+5+ versus BL.

Supplemental Table S5. The list of cell wall-modifying or -degradinggenes and their expression changes based on log2 fold.

Supplemental Table S6. The primers used in this study.

Supplemental Data Set 1. The differentially expressed genes in BL5+ com-pared with BL.

Supplemental Data Set 2. The differentially expressed genes in BL3+5+compared with BL.

ACKNOWLEDGMENTS

We thank Yihua Zhou (Institute of Genetics andDevelopmental Biology) forkindly providing the Pontamine Fast Scarlet 4B solution.

Received January 25, 2017; accepted May 4, 2017; published May 8, 2017.

LITERATURE CITED

Anderson CT, Carroll A, Akhmetova L, Somerville C (2010) Real-timeimaging of cellulose reorientation during cell wall expansion in Arabi-dopsis roots. Plant Physiol 152: 787–796

Bellincampi D, Cervone F, Lionetti V (2014) Plant cell wall dynamics andwall-related susceptibility in plant-pathogen interactions. Front PlantSci 5: 228

Bosch M, Cheung AY, Hepler PK (2005) Pectin methylesterase, a regulatorof pollen tube growth. Plant Physiol 138: 1334–1346

Brutus A, Sicilia F, Macone A, Cervone F, De Lorenzo G (2010) A domainswap approach reveals a role of the plant wall-associated kinase 1 (WAK1)as a receptor of oligogalacturonides. Proc Natl Acad Sci USA 107: 9452–9457

Cai YM, Yu J, Gallois P (2014) Endoplasmic reticulum stress-induced PCDand caspase-like activities involved. Front Plant Sci 5: 41

Carvalho HH, Silva PA, Mendes GC, Brustolini OJ, Pimenta MR, Gou-veia BC, Valente MA, Ramos HJ, Soares-Ramos JR, Fontes EP (2014)The endoplasmic reticulum binding protein BiP displays dual functionin modulating cell death events. Plant Physiol 164: 654–670

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

Chen J, Ding J, Ouyang Y, Du H, Yang J, Cheng K, Zhao J, Qiu S, ZhangX, Yao J, et al (2008) A triallelic system of S5 is a major regulator of thereproductive barrier and compatibility of indica-japonica hybrids in rice.Proc Natl Acad Sci USA 105: 11436–11441

Chen L, Ye D (2007) Roles of pectin methylesterases in pollen-tube growth.J Integr Plant Biol 49: 94–98

Choi D, Lee Y, Cho HT, Kende H (2003) Regulation of expansin gene ex-pression affects growth and development in transgenic rice plants. PlantCell 15: 1386–1398

Coimbra S, Jones B, Pereira LG (2008) Arabinogalactan proteins (AGPs)related to pollen tube guidance into the embryo sac in Arabidopsis. PlantSignal Behav 3: 455–456

Coolen S, Proietti S, Hickman R, Davila Olivas NH, Huang P-P, Van VerkMC, Van Pelt JA, Wittenberg AHJ, De Vos M, Prins M, et al (2016)Transcriptome dynamics of Arabidopsis during sequential biotic andabiotic stresses. Plant J 86: 249–267

Cosgrove DJ (1999) Enzymes and other agents that enhance cell wall ex-tensibility. Annu Rev Plant Physiol Plant Mol Biol 50: 391–417

1694 Plant Physiol. Vol. 174, 2017

Zhu et al.

https://plantphysiol.orgDownloaded on January 21, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.

Coyne J, Orr H (2004) Speciation. Sinauer Associates, Sunderland, MADe Block M, Debrouwer D (1993) RNA-RNA in situ hybridization using

digoxigenin-labeled probes: the use of high-molecular-weight polyvinylalcohol in the alkaline phosphatase indoxyl-nitroblue tetrazolium reac-tion. Anal Biochem 215: 86–89

Digel B, Pankin A, von Korff M (2015) Global transcriptome profiling ofdeveloping leaf and shoot apices reveals distinct genetic and environ-mental control of floral transition and inflorescence development inbarley. Plant Cell 27: 2318–2334

Dixon RA, Paiva NL (1995) Stress-induced phenylpropanoid metabolism.Plant Cell 7: 1085–1097

Dobzhansky T (1937) Genetics and the origin of species. Columbia Uni-versity Press, New York

Du H, Ouyang Y, Zhang C, Zhang Q (2011) Complex evolution of S5, amajor reproductive barrier regulator, in the cultivated rice Oryza sativaand its wild relatives. New Phytol 191: 275–287

Du Z, Zhou X, Ling Y, Zhang Z, Su Z (2010) agriGO: a GO analysis toolkitfor the agricultural community. Nucleic Acids Res 38: W64-70

Gao H, Zhang Y, Wang W, Zhao K, Liu C, Bai L, Li R, Guo Y (2017) Twomembrane-anchored aspartic proteases contribute to pollen and ovuledevelopment. Plant Physiol 173: 219–239

Gou JY, Miller LM, Hou G, Yu XH, Chen XY, Liu CJ (2012) Acetylesterase-mediated deacetylation of pectin impairs cell elongation, pollen germi-nation, and plant reproduction. Plant Cell 24: 50–65

Groszmann M, Gonzalez-Bayon R, Lyons RL, Greaves IK, Kazan K,Peacock WJ, Dennis ES (2015) Hormone-regulated defense and stressresponse networks contribute to heterosis in Arabidopsis F1 hybrids. ProcNatl Acad Sci USA 112: E6397–E6406

Guan B, Lei J, Su S, Chen F, Duan Z, Chen Y, Gong X, Li H, Jin J (2012)Absence of Yps7p, a putative glycosylphosphatidylinositol-linked as-partyl protease in Pichia pastoris, results in aberrant cell wall composi-tion and increased osmotic stress resistance. FEMS Yeast Res 12: 969–979

Guo K, Zou W, Feng Y, Zhang M, Zhang J, Tu F, Xie G, Wang L, Wang Y,Klie S, et al (2014) An integrated genomic and metabolomic frameworkfor cell wall biology in rice. BMC Genomics 15: 596

Hamann T (2012) Plant cell wall integrity maintenance as an essentialcomponent of biotic stress response mechanisms. Front Plant Sci 3: 77

Hara Y, Yokoyama R, Osakabe K, Toki S, Nishitani K (2014) Function ofxyloglucan endotransglucosylase/hydrolases in rice. Ann Bot (Lond)114: 1309–1318

Hayashi S, Takahashi H, Wakasa Y, Kawakatsu T, Takaiwa F (2013)Identification of a cis-element that mediates multiple pathways of theendoplasmic reticulum stress response in rice. Plant J 74: 248–257

Hayashi S, Wakasa Y, Takahashi H, Kawakatsu T, Takaiwa F (2012)Signal transduction by IRE1-mediated splicing of bZIP50 and otherstress sensors in the endoplasmic reticulum stress response of rice. PlantJ 69: 946–956

Hepler PK, Rounds CM, Winship LJ (2013) Control of cell wall extensi-bility during pollen tube growth. Mol Plant 6: 998–1017

Howell SH (2013) Endoplasmic reticulum stress responses in plants. AnnuRev Plant Biol 64: 477–499

Hu K, Cao J, Zhang J, Xia F, Ke Y, Zhang H, Xie W, Liu H, Cui Y, Cao Y,et al (2017) Improvement of multiple agronomic traits by a disease re-sistance gene via cell wall reinforcement. Nat Plants 3: 17009

Ikehashi H, Araki H (1986) Genetics of F1 sterility in remote crosses of rice.In IRRI, SJ Banta, GS Argosino, eds. Rice Genetics. IRRI, Manila, pp 119–130

Kawahara Y, de la Bastide M, Hamilton JP, Kanamori H, McCombie WR,Ouyang S, Schwartz DC, Tanaka T, et al (2013) Improvement of theOryza sativa Nipponbare reference genome using next generation se-quence and optical map data. Rice (NY) 6: 4

Kourmpetli S, Lee K, Hemsley R, Rossignol P, Papageorgiou T, Drea S(2013) Bidirectional promoters in seed development and related hor-mone/stress responses. BMC Plant Biol 13: 187

Krysan DJ (2009) The cell wall and endoplasmic reticulum stress responsesare coordinately regulated in Saccharomyces cerevisiae. Commun IntegrBiol 2: 233–235

Krysan DJ, Ting EL, Abeijon C, Kroos L, Fuller RS (2005) Yapsins are afamily of aspartyl proteases required for cell wall integrity in Saccharo-myces cerevisiae. Eukaryot Cell 4: 1364–1374

Kubo T, Takashi T, Ashikari M, Yoshimura A, Kurata N (2016) Twotightly linked genes at the hsa1 locus cause both F1 and F2 hybrid sterilityin rice. Mol Plant 9: 221–232

Liu A, Zhang Q, Li H (1992) Location of a gene for wide-compatibility inthe RFLP linkage map. Rice Genet Newsl 9: 134–136

Liu K, Wang J, Li H, Xu C, Liu A, Li X, Zhang Q (1997) A genome-wideanalysis of wide compatibility in rice and the precise location of the S5locus in the molecular map. Theor Appl Genet 95: 809–814

Long Y, Zhao L, Niu B, Su J, Wu H, Chen Y, Zhang Q, Guo J, Zhuang C,Mei M, et al (2008) Hybrid male sterility in rice controlled by interactionbetween divergent alleles of two adjacent genes. Proc Natl Acad Sci USA105: 18871–18876

Ma N, Wang Y, Qiu S, Kang Z, Che S, Wang G, Huang J (2013) Over-expression of OsEXPA8, a root-specific gene, improves rice growth androot system architecture by facilitating cell extension. PLoS One 8: e75997

Maris A, Suslov D, Fry SC, Verbelen JP, Vissenberg K (2009) Enzymiccharacterization of two recombinant xyloglucan endotransglucosylase/hydrolase (XTH) proteins of Arabidopsis and their effect on root growthand cell wall extension. J Exp Bot 60: 3959–3972

Miedes E, Suslov D, Vandenbussche F, Kenobi K, Ivakov A, Van DerStraeten D, Lorences EP, Mellerowicz EJ, Verbelen JP, Vissenberg K(2013) Xyloglucan endotransglucosylase/hydrolase (XTH) overexpressionaffects growth and cell wall mechanics in etiolated Arabidopsis hypocotyls. JExp Bot 64: 2481–2497

Mizuta Y, Harushima Y, Kurata N (2010) Rice pollen hybrid incompati-bility caused by reciprocal gene loss of duplicated genes. Proc Natl AcadSci USA 107: 20417–20422

Muller HJ (1942) Isolating mechanisms, evolution, and temperature. BiolSymp 6: 71–125

Niu N, Liang W, Yang X, Jin W, Wilson ZA, Hu J, Zhang D (2013) EAT1promotes tapetal cell death by regulating aspartic proteases during malereproductive development in rice. Nat Commun 4: 1445

Ouyang Y, Chen J, Ding J, Zhang Q (2009) Advances in the understandingof inter-subspecific hybrid sterility and wide-compatibility in rice. ChinSci Bull 54: 2332–2341

Ouyang Y, Li G, Mi J, Xu C, Du H, Zhang C, Xie W, Li X, Xiao J, Song H,et al (2016) Origination and establishment of a trigenic reproductiveisolation system in rice. Mol Plant 9: 1542–1545

Ouyang Y, Zhang Q (2013) Understanding reproductive isolation based onthe rice model. Annu Rev Plant Biol 64: 111–135

Paparella S, Tava A, Avato P, Biazzi E, Macovei A, Biggiogera M, CarboneraD, Balestrazzi A (2015) Cell wall integrity, genotoxic injury and PCD dy-namics in alfalfa saponin-treated white poplar cells highlight a complex linkbetween molecule structure and activity. Phytochemistry 111: 114–123

Pirselova B, Matusikova I (2012) Callose: the plant cell wall polysaccharidewith multiple biological functions. Acta Physiol Plant 35: 635–644

Pogorelko G, Lionetti V, Bellincampi D, Zabotina O (2013) Cell wall in-tegrity: targeted post-synthetic modifications to reveal its role in plantgrowth and defense against pathogens. Plant Signal Behav 8: e25435

Qiu SQ, Liu K, Jiang JX, Song X, Xu CG, Li XH, Zhang Q (2005) Delim-itation of the rice wide compatibility gene S5 ( n ) to a 40-kb DNAfragment. Theor Appl Genet 111: 1080–1086

Ramsak �Z, Baebler Š, Rotter A, Korbar M, Mozetic I, Usadel B, Gruden K(2014) GoMapMan: integration, consolidation and visualization of plantgene annotations within the MapMan ontology. Nucleic Acids Res 42:D1167–D1175

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 wallproteins by Candida albicans Sap9 and Sap10. Eukaryot Cell 10: 98–109

Scrimale T, Didone L, de Mesy Bentley KL, Krysan DJ (2009) The un-folded protein response is induced by the cell wall integrity mitogen-activated protein kinase signaling cascade and is required for cell wallintegrity in Saccharomyces cerevisiae. Mol Biol Cell 20: 164–175

Seehausen O, Butlin RK, Keller I, Wagner CE, Boughman JW, HohenlohePA, Peichel CL, Saetre G-P, Bank C, Brännström A, et al (2014) Ge-nomics and the origin of species. Nat Rev Genet 15: 176–192

Song X, Qiu SQ, Xu CG, Li XH, Zhang Q (2005) Genetic dissection of em-bryo sac fertility, pollen fertility, and their contributions to spikelet fer-tility of intersubspecific hybrids in rice. Theor Appl Genet 110: 205–211

Takahashi H, Wang S, Hayashi S, Wakasa Y, Takaiwa F (2014) Cis-elementof the rice PDIL2-3 promoter is responsible for inducing the endoplasmicreticulum stress response. J Biosci Bioeng 117: 620–623

Tanaka K, Murata K, Yamazaki M, Onosato K, Miyao A, Hirochika H(2003) Three distinct rice cellulose synthase catalytic subunit genes re-quired for cellulose synthesis in the secondary wall. Plant Physiol 133:73–83

Plant Physiol. Vol. 174, 2017 1695

Transcriptomic Basis of a Rice Reproductive Barrier

https://plantphysiol.orgDownloaded on January 21, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.

Thimm O, Bläsing O, Gibon Y, Nagel A, Meyer S, Krüger P, Selbig J,Müller LA, Rhee SY, Stitt M (2004) MAPMAN: a user-driven tool todisplay genomics data sets onto diagrams of metabolic pathways andother biological processes. Plant J 37: 914–939

Tholl D (2006) Terpene synthases and the regulation, diversity and bio-logical roles of terpene metabolism. Curr Opin Plant Biol 9: 297–304

Thomas J, Ingerfeld M, Nair H, Chauhan SS, Collings DA (2012) Pont-amine fast scarlet 4B: a new fluorescent dye for visualising cell wallorganisation in radiata pine tracheids. Wood Sci Technol 47: 59–75

Trapnell C, Roberts A, Goff L, Pertea G, Kim D, Kelley DR, Pimentel H,Salzberg SL, Rinn JL, Pachter L (2012) Differential gene and transcriptexpression analysis of RNA-seq experiments with TopHat and Cufflinks.Nat Protoc 7: 562–578

Ünal M, Vardar F, Aytürk Ö (2013) Callose in plant sexual reproduction.In M Silva-Opps, ed, Current Progress in Biological Research. InTech,Croatia, pp 319–343

Vogel JT, Zarka DG, Van Buskirk HA, Fowler SG, Thomashow MF (2005)Roles of the CBF2 and ZAT12 transcription factors in configuring thelow temperature transcriptome of Arabidopsis. Plant J 41: 195–211

Wakasa Y, Oono Y, Yazawa T, Hayashi S, Ozawa K, Handa H, Matsumoto T,Takaiwa F (2014) RNA sequencing-mediated transcriptome analysis of riceplants in endoplasmic reticulum stress conditions. BMC Plant Biol 14: 101

Wan S, Jiang L (2016) Erratum to: Endoplasmic reticulum (ER) stress andthe unfolded protein response (UPR) in plants. Protoplasma 253: 765

Wang GW, He YQ, Xu CG, Zhang Q (2005) Identification and confirmationof three neutral alleles conferring wide compatibility in inter-subspecifichybrids of rice (Oryza sativa L.) using near-isogenic lines. Theor ApplGenet 111: 702–710

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 ApplGenet 97: 407–412

WangQ,Wan L, Li D, Zhu L, QianM, DengM (2009) Searching for bidirectionalpromoters in Arabidopsis thaliana. BMC Bioinformatics (Suppl) 10: S29

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 regulatorof growth, development, and stress response. Plant Physiol 164: 735–747

Winkel-Shirley B (2002) Biosynthesis of flavonoids and effects of stress.Curr Opin Plant Biol 5: 218–223

Xiao C, Somerville C, Anderson CT (2014) POLYGALACTURONASE IN-VOLVED IN EXPANSION1 functions in cell elongation and flower de-velopment in Arabidopsis. Plant Cell 26: 1018–1035

Xu H, Xu W, Xi H, Ma W, He Z, Ma M (2013) The ER luminal bindingprotein (BiP) alleviates Cd(2+)-induced programmed cell death throughendoplasmic reticulum stress-cell death signaling pathway in tobaccocells. J Plant Physiol 170: 1434–1441

Yamagata Y, Yamamoto E, Aya K, Win KT, Doi K, Sobrizal, Ito T, KanamoriH, Wu J, Matsumoto T, et al (2010) Mitochondrial gene in the nuclear ge-nome induces reproductive barrier in rice. Proc Natl Acad Sci USA 107:1494–1499

Yang J, Sardar HS, McGovern KR, Zhang Y, Showalter AM (2007) Alysine-rich arabinogalactan protein in Arabidopsis is essential for plantgrowth and development, including cell division and expansion. Plant J49: 629–640

Yang J, Zhao X, Cheng K, Du H, Ouyang Y, Chen J, Qiu S, Huang J, JiangY, Jiang L, et al (2012) A killer-protector system regulates both hybridsterility and segregation distortion in rice. Science 337: 1336–1340

Yu Y, Zhao Z, Shi Y, Tian H, Liu L, Bian X, Xu Y, Zheng X, Gan L, Shen Y,et al (2016) Hybrid sterility in rice (Oryza sativa L.) involves the tetra-tricopeptide repeat domain containing protein. Genetics 203: 1439–1451

Zhang GY, Feng J, Wu J, Wang XW (2010) BoPMEI1, a pollen-specific pectinmethylesterase inhibitor, has an essential role in pollen tube growth.Planta 231: 1323–1334

Zhang JW, Xu L, Wu YR, Chen XA, Liu Y, Zhu SH, Ding WN, Wu P, YiKK (2012) OsGLU3, a putative membrane-bound endo-1,4-beta-glucanase, is required for root cell elongation and division in rice (Or-yza sativa L.). Mol Plant 5: 176–186

ZhiMing Y, Bo K, XiaoWei H, ShaoLei L, YouHuang B, WoNa D, Ming C,Hyung-Taeg C, Ping W (2011) Root hair-specific expansins modulateroot hair elongation in rice. Plant J 66: 725–734

Zhong R, Lee C, McCarthy RL, Reeves CK, Jones EG, Ye ZH (2011)Transcriptional activation of secondary wall biosynthesis by rice andmaize NAC and MYB transcription factors. Plant Cell Physiol 52: 1856–1871

Zhou HL, He SJ, Cao YR, Chen T, Du BX, Chu CC, Zhang JS, Chen SY(2006) OsGLU1, a putative membrane-bound endo-1,4-beta-D-glucanasefrom rice, affects plant internode elongation. Plant Mol Biol 60: 137–151

1696 Plant Physiol. Vol. 174, 2017

Zhu et al.

https://plantphysiol.orgDownloaded on January 21, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.