Reproductive barriers in indica-japonica rice hybrids ...

Plant Physiology Preview. Published on May 8, 2017, as DOI:10.1104/pp.17.00093

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Running Head:

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Transcriptomic basis of a rice reproductive barrier

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Corresponding Author:

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Qifa Zhang

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National Key Laboratory of Crop Genetic Improvement and National Center of Plant

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Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China

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Phone: 86-27-87282429

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Fax: 86-27-87287092

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E-mail: [email protected]

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Research Area:

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Genes, Development and Evolution

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1 Downloaded from www.plantphysiol.org on May 27, 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

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Title of article:

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Reproductive barriers in indica-japonica rice hybrids are uncovered by transcriptome

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analysis

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All authors’ full names:

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Yanfen Zhu, Yiming Yu, Ke Cheng, Yidan Ouyang, Jia Wang, Liang Gong, Qinghua

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Zhang, Xianghua Li, Jinghua Xiao and Qifa Zhang*

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*For correspondence (Fax: 86-27-87287092; Phone: 86-27-87282429; e-mail:

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[email protected])

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Author contributions:

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Y.Z. and Qifa Zhang designed the research; Y.Z., Y.Y., K.C., Y.O., J.W., L.G. and

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Qinghua Zhang performed the research; X.L. and J.X. contributed reagents; Y.Z. and

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Qifa Zhang analyzed data and wrote the paper.

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The authors declare no conflict of interests.

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Affiliations:

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National Key Laboratory of Crop Genetic Improvement and National Center of Plant

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Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China

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One Sentence Summary:

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A reproductive barrier causes female gamete abortion of indica-japonica rice hybrids

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by damaging cell wall integrity inducing massive biotic and abiotic stresses resulting

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in ER stress leading to premature PCD.

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Footnotes:

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Financial source: This work was supported by grants from the National Key Research

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and Development Program (2016YFD0100903) and National Natural Science

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Foundation (31130032) of China.

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ABSTRACT

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In rice (Oryza sativa L.), hybrids between indica and japonica subspecies are usually

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highly sterile, which provides a model system for studying postzygotic reproductive

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isolation. A killer-protector system S5 composed of three adjacent genes, ORF3,

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ORF4 and ORF5, regulates female gamete fertility of indica-japonica hybrids. To

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characterize the processes underlying this system, we performed transcriptomic

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analyses of pistils from rice variety Balilla (BL), Balilla with transformed ORF5+

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(BL5+) producing sterile female gametes, and Balilla with transformed ORF3+ and

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ORF5+ (BL3+5+) producing fertile gametes. RNA sequencing of tissues collected

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before (MMC), during (MEI), and after (AME) meiosis of the megaspore mother cell

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detected 19,269 to 20,928 genes as expressed. Comparison between BL5+ and BL

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showed that ORF5+ induced differential expression of 8,339, 6,278 and 530 genes at

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MMC, MEI, and AME. At MMC, large-scale differential expression of cell wall

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modifying genes and biotic and abiotic response genes indicated that cell wall

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integrity damage induced severe biotic and abiotic stresses. The processes continued

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to MEI and induced ER stress as indicated by differential expression of ER stress

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responsive genes, leading to PCD at MEI and AME, resulting in abortive female

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gametes. In the BL3+5+/BL comparison, 3,986, 749 and 370 genes were

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differentially expressed at MMC, MEI and AME. Large numbers of cell wall

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modification and biotic and abiotic response genes were also induced at MMC but

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largely suppressed at MEI without inducing ER stress and PCD, producing fertile

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gametes. These results have general implications for the understanding of biological

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processes underlying reproductive barriers.

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INTRODUCTION

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Reproductive isolation, a phenomenon widely existing in natural organisms,

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promotes speciation and maintains the integrity of species over time (Coyne and Orr,

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2004). It is divided into two general categories depending on whether it occurs before

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or after fertilization: prezygotic reproductive isolation and postzygotic reproductive

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isolation (Seehausen et al., 2014). The former prevents the formation of hybrid

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zygotes, while the latter results in hybrid incompatibility including hybrid necrosis,

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weakness, sterility, and lethality in F1 or later generations (Ouyang and Zhang, 2013).

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The Dobzhansky-Muller model suggests that hybrid incompatibility is caused by the

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negative interactions between functionally divergent genes in the parental species

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(Dobzhansky, 1937; Muller, 1942).

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Rice, a major food crop and model plant for genomic study of monocotyledon

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species, also provides a model system for studying reproductive isolation. The Asian

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cultivated rice is composed of two subspecies, indica and japonica. Their hybrids are

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usually highly sterile, which is a barrier for utilization of the strong vigor in

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indica-japonica hybrids. A number of loci responsible for hybrid sterility have been

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identified, including ones that cause embryo-sac sterility, pollen sterility and spikelet

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sterility (Ouyang et al., 2009). So far, six loci responsible for hybrid incompatibility

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have been cloned. Among them, DPL1/DPL2 (Mizuta et al., 2010), S27/S28

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(Yamagata et al., 2010) and Sa (Long et al., 2008) are responsible for pollen fertility,

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while hsa1 (Kubo et al., 2016), S7 (Yu et al., 2016), and S5 (Chen et al., 2008; Yang et

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al., 2012) control female fertility, all of them cause spikelet sterility. Three

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evolutionary genetic models have been proposed to summarize the findings: parallel

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divergence model, sequential divergence model and parallel-sequential divergence

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model (Ouyang and Zhang, 2013). DPL1/DPL2 and S27/S28 in rice, HPA1/HPA2 in

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Arabidopsis, and JYAlpha in Drosophila conform to the parallel divergence model. Sa,

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hsa1 and many loci in other species can be explained by the sequential divergence

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model. However, S5 in rice is the only case identified so far to follow the

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parallel-sequential divergence model.

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S5 is a major locus controlling indica-japonica hybrid sterility identified in many 4 Downloaded from www.plantphysiol.org on May 27, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.

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studies (Ikehashi and Araki, 1986; Liu et al., 1992; Liu et al., 1997; Wang et al., 1998;

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Qiu et al., 2005; Song et al., 2005; Wang et al., 2005; Chen et al., 2008), and is also

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associated with the divergence and domestication of rice (Du et al., 2011; Ouyang et

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al., 2016). In a typical indica-japonica hybrid, the preferential abortion of female

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gametes with japonica allele is regulated cooperatively by three adjacent genes, ORF3,

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ORF4, and ORF5 at the S5 locus (Yang et al., 2012). Typical japonica and indica

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varieties carry ORF3-ORF4+ORF5- and ORF3+ORF4-ORF5+, respectively, where

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the functional allele of each gene is indicated with “+” while the non-functional allele

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as “-”. ORF5 encodes an aspartic protease with cell wall localization. The indica

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allele ORF5+ differs from the japonica allele ORF5- by two nucleotides, both of

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which cause amino acid changes (Chen et al., 2008). The japonica allele ORF4+

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encodes an unknown transmembrane protein while the indica allele ORF4- has an

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11-bp deletion resulting in an endoplasmic reticulum (ER)-localization. ORF3 is

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predicted to encode a heat shock protein (HSP) resident in ER functioning to resolve

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ER stress. The japonica allele ORF3- has a frameshift in the C terminus relative to the

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indica ORF3+ due to a 13-bp deletion. In this system, extracellular ORF5+

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presumably catalyzes its substrate producing a substance that is sensed by the

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transmembrane protein ORF4+, which induces ER stress and subsequently leading to

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premature programed cell death (PCD). It is speculated that ORF3+, but not ORF3-,

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can prevent/resolve the ER stress caused by ORF5+/ORF4+. Based on the results of

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genetic and cytological analyses, Yang et al (2012) proposed a killer-protector model

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for the roles of these three genes in this system: ORF5+, together with ORF4+, kills

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the gametes by inducing ER stress that causes premature PCD. ORF3+ protects the

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gametes from being killed thus restores the hybrids fertility by preventing/resolving

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ER stress.

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In recent years, transcriptomic analysis based on RNA-sequencing technology

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has become a powerful tool to characterize regulatory networks and pathways to

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enhance understanding of biological processes (Vogel et al., 2005; Digel et al., 2015;

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Groszmann et al., 2015; Coolen et al., 2016). In this study, we performed a

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comparative transcriptomic analysis of pistils from Balilla (a typical japonica variety), 5 Downloaded from www.plantphysiol.org on May 27, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.

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BalillaORF5+ (Balilla with a transformed ORF5+) and BalillaORF3+ORF5+

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(Balilla with transformed ORF3+ and ORF5+) before, during, and after the

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megaspore meiosis. The comparisons detected thousands of genes that were

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differentially regulated in these three genotypes at various stages. Functional

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classification of the differentially regulated genes identified major patterns of

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differential expression, which suggested the biological processes underlying the

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killer-protector system of the S5 locus. These results provide insights into the

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functional roles of ORF3+, ORF4+ and ORF5+ genes as well as the cellular and

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biochemical nature of their interactions in regulating indica-japonica hybrid sterility.

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RESULTS

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Ovule abortion process of BL5+

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Previous study (Yang et al., 2012) showed that the S5 reproductive barrier is

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composed of three tightly-linked genes, ORF3, ORF4 and ORF5, in which ORF5+

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and ORF4+ together function as the killer of the female gametes and ORF3+ protects

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the gametes from killing. A typical indica variety has the haplotype of ORF3+,

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ORF4-, ORF5+ and japonica variety is of ORF3-, ORF4+, ORF5-. Balilla

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(abbreviated BL) is a typical japonica variety. Transgenic plants BalillaORF5+

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(abbreviated BL5+) carrying a transformed ORF5+ under its native promoter showed

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extremely low spikelet fertility (6.50%) compared with wild type BL (75.52%). In

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contrast, BalillaORF3+ORF5+ (abbreviated BL3+5+), carrying both transformed

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ORF5+ and transformed ORF3+ (also under its native promoter), had normal spikelet

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fertility (77.35%) due to the protective role of ORF3+ (Supplemental Fig. S1A-C).

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Paraffin section results showed that the ovule development process in both BL and

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BL3+5+ was normal. In brief, the archesporial cell enlarged initially and developed

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into a megaspore mother cell (Fig. 1A, K). The megaspore mother cell then

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underwent meiotic division forming four megaspores in a line (Fig. 1B, C, L, and M).

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The three megaspores near the micropyle degenerated, while the megaspore nearest

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the chalaza enlarged and developed into a functional megaspore (FM) (Fig. 1D, N).

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The FM underwent three rounds of mitotic division to produce an eight-nucleate

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embryo-sac (Fig. 1E, O). In the aborted ovule of BL5+, the megaspore mother cell,

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dyad and tetrad could be produced as in BL (Fig. 1F-H). However, the nucellus cells

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showed vacuolization and even degeneration at the meiosis stage (Fig. 1G-H). After

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meiosis, morphological abnormalities of both nucellus cells and FM were observed

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(Fig. 1I). The abnormal FM did not enlarge and failed to develop into a mature

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embryo-sac. The aborted embryo-sac accumulated unknown substances that were

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deeply stained (Fig. 1J). Thus, the megaspore meiosis process in BL5+ was normal

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but the nucellus cells around the megaspores showed serious defects at the megaspore

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meiosis stage. The nucellus cells degradation and FM abortion caused the sterility of

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BL5+. 7 Downloaded from www.plantphysiol.org on May 27, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.

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ORF5 and ORF4 were neighboring genes overlapping in their promoter region

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(Yang et al., 2012). This kind of gene pairs are frequently co-expressed (Wang et al.,

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2009; Kourmpetli et al., 2013). ORF5 and ORF4 had similar expression profile and

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relative high expression in reproductive organs (Yang et al., 2012). The in situ

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hybridization showed that ORF5+ had localized expression in developing ovules

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(Chen et al., 2008). Similarly, ORF4+ also displayed high and specific expression in

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ovule tissues (Supplemental Fig. S2G-K). The specific expression of ORF5+ and

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ORF4+ in ovules was in accordance with the specific abortion of ovules in BL5+.

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However, the protector ORF3+ showed a wide range of expression in many tissues

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including ovules, indicating its possible function in many processes (Supplemental

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Fig. S2A-F).

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Based on the ovule development process, we divided the ovule abortion process

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into three stages (Fig. 1). The first was the megaspore mother cell (MMC) stage when

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the ovule had megasporocyte and no obvious abnormalities were observed in BL5+. 8 Downloaded from www.plantphysiol.org on May 27, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.

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The second was the meiosis (MEI) stage when meiosis was in progress and the

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nucellus cells of BL5+ were vacuolated. The third was after meiosis (AME) when

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some unknown substances accumulated in embryo-sac (Fig. 1).

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Processes underlying gamete abortion in BL5+

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We collected the pistils of wild type BL, sterile BL5+ and fertility-restored

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BL3+5+ at MMC, MEI and AME, and performed transcriptomic analysis. A data

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matrix of three stages from three genotypes was generated. Totally 19,269 to 20,928

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genes were detected as expressed in these tissues at different stages representing

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34.53% to 37.50% of total annotated genes of the Nipponbare reference genome

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(Supplemental Table S1) (Kawahara et al., 2013). A total of 8,339, 6,278 and 530

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genes were differentially expressed in BL5+ compared with BL at MMC, MEI and

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AME, respectively, including 4,234, 2,430 and 410 genes that were up-regulated at

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the three stages and 4,105, 3,848 and 120 genes that were down-regulated

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(Supplemental Table S2; Supplemental Dataset 1). Among the differentially expressed

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genes (DEGs), 2,067 were up-regulated and 3,325 were down-regulated at both MMC

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and MEI (Fig. 2A). These 5,392 common DEGs accounted for 64.66% and 85.89% of

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total DEGs at MMC and MEI, respectively, indicating that MMC and MEI involved

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substantially overlapping cellular and molecular responses.

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An overview of the differentially induced processes in BL5+ revealed by the

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transcriptomes. We performed MapMan and Gene Ontology (GO) enrichment

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analyses to classify the DEGs. MapMan is an effective tool to analyze metabolic

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pathways and other biological processes of the transcriptome systematically (Thimm

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et al., 2004; Ramsak et al., 2014). To get a global view of the 8,339 DEGs in BL5+ at

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MMC, we used the “Metabolism overview” and “Cellular response” of MapMan to

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outline the transcriptional changes of genes with putative functions in metabolism and

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cellular response (Fig. 2B-C).

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In “Metabolism overview”, primary metabolic processes, including cell wall (354

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genes), amino acid (226 genes), lipids (316 genes), nucleotide (81 genes) and major

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carbohydrate (128 genes) metabolism, accounted for 13.25% of total DEGs. The cell 9 Downloaded from www.plantphysiol.org on May 27, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.

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wall localization of ORF5+, which was previously hypothesized to initiate the

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premature PCD (Yang et al., 2012), led us to focus our attention to the cell wall

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metabolic genes. We found that 187 (128 up-regulated and 59 down-regulated) of the

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354 genes were involved in cell wall degradation and modification, which may affect

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the cell wall structures in one way or another. The secondary metabolism processes

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including terpenes (64 genes), flavonoids (105 genes), and phenylpropanoids (120

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genes) metabolism, which are important in plant stress response (Dixon and Paiva,

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1995; Winkelshirley, 2002; Tholl, 2006), constituted 3.47% of all DEGs. In the term


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of “Cellular response”, 8.65% (721 genes) of DEGs were responsive to biotic and

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abiotic stress; 1.94% (162 genes) of DEGs were related to redox, a phenomenon

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usually triggered by stress; 6.67% (667 genes) of DEGs functioned in developmental

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processes.

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GO analysis for up-regulated genes revealed that three GO terms related to cell

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wall metabolism, seven GO terms involved in stress or stimulus response, and eleven

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GO terms associated with gene expression, protein synthesis and translation were

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significantly enriched (FDR < 0.005) (Fig. 3).

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The change in cell wall structure by cell wall modification genes may cause cell

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wall integrity damage, which was similar to the situation of pathogen or pest attack.

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We thus explored the “Biotic stress” in BL5+ at MMC and found that DEGs in

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various functional categories of biotic stress were enriched including R genes,

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pathogen-related proteins, signaling, hormone signaling, proteolysis, ROS-regulation

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and biotic stress-responsive transcription factors (Supplemental Fig. S3A).

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Thus, both of the MapMan and GO results indicated that a severe stress response

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including both biotic and abiotic stresses were induced in BL5+ at MMC, which may

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be caused by the damage in cell wall integrity, even though no abnormalities were

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observed at this stage (Fig. 1F).

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At MEI, 163 of the 300 DEGs in “cell wall” were related to the cell wall

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degradation and modification including 102 up-regulated and 61 down-regulated ones

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(Fig. 2B-C). Cell wall-related GO terms such as “glucan metabolic process”,

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“polysaccharide metabolic process” and “polysaccharide biosynthetic process” were

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enriched (Fig. 3). The continuous up-regulation of cell wall degrading and modifying

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genes at MMC and MEI might be related to the observed cell wall abnormalities such

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as vacuolization and degradation at MEI (Fig. 1G-H). In addition, 599 DEGs (9.54%

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of total DEGs) in “biotic stress” and “abiotic stress” and genes in various functional

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categories of biotic stress were identified (Fig. 2B, Supplemental Fig. S3B),

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suggesting that the stress response also continued at MEI.

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By the AME stage, DEG numbers in all the metabolic processes and cellular

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responses were largely reduced compared to those at MMC and MEI (Fig. 2B). The 11 Downloaded from www.plantphysiol.org on May 27, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.

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ovule had aborted with the accumulation of unknown substances. In contrast, however,

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a GO term “cellulose biosynthetic process” was significantly enriched (Fig. 3),

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indicating misregulation of cellulose biosynthesis in BL5+. 12 Downloaded from www.plantphysiol.org on May 27, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.

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Large-scale transcriptional changes of cell wall modifying and degrading

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genes in BL5+ at MMC and MEI. The MapMan and GO results showed that cell

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wall metabolism especially cell wall modification and degradation were active in

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BL5+ (Fig. 2). Cell wall modifying and degrading genes, also called cell wall

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remodeling genes, affect cell growth by changing the extensibility and

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physiochemical properties of the cell wall (Cosgrove, 1999; Yu et al., 2011; Gou et al.,

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2012; Hepler et al., 2013; Xiao et al., 2014). We analyzed the expression of genes

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from

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endotransglucosylase/hydrolases (XTHs), pectin methyl esterases (PMEs), pectin

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acetylesterases (PAEs), polygalacturonases (PGases), glycoside hydrolase family 9

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(GH9) and arabinogalactan proteins (AGPs), which affect cell wall structure and

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extension by enzymatic or non-enzymatic ways (Bosch et al., 2005; Coimbra et al.,

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2008; Maris et al., 2009; Gou et al., 2012; Che et al., 2016). We found that 95, 85 and

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23 genes from these families were differentially expressed at MMC, MEI, and AME,

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respectively, of which 73 genes were differentially expressed at both MMC and MEI,

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including 59 up-regulated and 14 down-regulated (Fig. 4A-B). The 59 up-regulated

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genes consisted of 13 EXPs, 7 XTHs, 5 PMEs, 4 PAEs, 8 PGases, 7 GH9s and 15

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AGPs. OsEXP4 was elevated in BL5+ at MMC and MEI. In rice, overexpression of

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OsEXP4 results in increased cell length, while repression of OsEXP4 reduces cell

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length (Choi et al., 2003). Three up-regulated XTHs (Os06g48160, Os03g01800 and

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Os10g39840) have been characterized to utilize xyloglucan, one of the cell wall

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components, as substrate (Hara et al., 2014). OsGLU1, a GH9 member, which

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regulates cell elongation in rice (Zhou et al., 2006; Zhang et al., 2012), was found

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up-regulated as well. The up-regulation of genes involved in cell wall degradation and

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cell size regulation was in accordance with cell wall abnormalities in BL5+ (Fig.

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1G-H). Thus, we inferred that the up-regulation of the cell wall remodeling genes

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induced by ORF5+ in BL5+ led to cell structure change, damaging the cell wall

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integrity.

seven

main

enriched

families:

expansins

(EXPs),

xyloglucan

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Cellulose deposition in BL5+. In BL5+, unknown substances were accumulated

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in the aborted ovule at AME (Fig. 1I-J). The enrichment of GO term “cellulose 13 Downloaded from www.plantphysiol.org on May 27, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.

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biosynthetic process” suggested a misregulation of cellulose biosynthesis and that the

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deposited substances in the aborted ovule might over-accumulate cellulose. To

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validate this, we used Pontamine fast scarlet 4B (S4B) and calcofluor white to stain

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cellulose (Anderson et al., 2010; Thomas et al., 2012), and observed strong

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fluorescence signals in BL5+ at the site where the substances accumulated, indicating

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that cellulose was indeed in large quantity (Fig. 5B, E), which was also supported by

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the much higher cellulose content in panicles of BL5+ than in BL (Fig. 5G). In

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contrast, normal mature embryo-sac structures were easily identified in the ovule of

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BL and BL3+5+, where low fluorescence intensity was detected (Fig. 5A, C, D, and

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F).

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Cellulose is a major cell wall polysaccharide composed of unbranched β-1,

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4-linked glucan chains. In rice, OsMYB46 and OsSWNs are secondary cell wall


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biosynthetic regulators. OsSWN1 acts on the upstream of OsMYB46 by binding to the

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SNBE element in its promoter region. Overexpression of OsSWN1 or OsMYB46 in

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Arabidopsis results in the ectopic deposition of cellulose, xylan and lignin (Zhong et

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al., 2011). In BL5+, OsSWN1, OsSWN4, OsSWN6 and OsMYB46 were up-regulated

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at MMC and MEI. Three cellulose synthases, OsCESA4, OsCESA7 and OsCESA9,

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which are regarded as subunits of cellulose synthase complex (Tanaka, 2003), were

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continuously up-regulated from MMC to AME. In addition, expression of five

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cellulose synthase-like genes (OsCslA1, OsCslA3, OsCslA5, OsCslA9 and OsCslF6)

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and two brittle culm genes (BC1 and BC3) were also elevated at MMC or MEI (Fig.

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4C). Upstream transcription factors such as OsSWN1, OsSWN6 and OsMYB46 had

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peak expression at MEI while their downstream genes such as OsCESAs and two

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brittle culm genes had highest expression at AME (Supplemental Fig. S4), in

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agreement with the over-accumulation of cellulose at AME (Fig. 5B, E). These results

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showed that the cellulose biosynthetic pathway was highly activated in BL5+

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resulting in the accumulation of cellulose in the aborted embryo-sac.

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Callose deposition in BL5+. Callose is a dynamic component of plant cell wall,

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which plays important roles in megasporogenesis. The uneven distribution of the

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callose wall around the megaspores decides the polarization of megaspores, the


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formation of a functional megaspore and the abortion of the other three non-functional

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megaspores (Ünal et al., 2013). Since many genes encoding β-1,3-glucanases, which

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have putative functions in callose metabolism, were differentially expressed in BL5+

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at MMC and MEI (Supplemental Fig. S5), we investigated the dynamic callose

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distributions in ovule of BL, BL5+ and BL3+5+. During meiosis, callose

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preferentially accumulated at the micropylar end of megaspores. Two callose bands

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were observed in all three genotypes at the dyad stage (Fig. 6A-F), and at tetrad stage

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at least three callose bands could be recognized also in all three genotypes although

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slightly different in distribution patterns (Fig. 6G-L). This apparent normal callose

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distribution in megaspores of BL5+ indicated likely normal meiosis. Unlike BL and

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BL3+5+, however, weak callose deposition was detected in nucellus cells at the

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tetrad stage in BL5+ (Fig. 6I). At the functional megaspore (FM) stage, callose

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accumulated in the three degenerated gametes but not in the FM (Fig. 6M-R). The

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survived FM subsequently developed to embryo-sac (Fig. 6S, T, W, X). However, the 16 Downloaded from www.plantphysiol.org on May 27, 2017 - Published by www.plantphysiol.org Copyright © 2017 American Society of Plant Biologists. All rights reserved.

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ovule of BL5+ displayed irregular callose deposition around the FM and in nucellus

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cells (Fig. 6O, P), and finally, the aborted embryo-sac was stuffed with large amount

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of callose (Fig. 6U, V). Thus, ORF5+ resulted in a misregulation of callose deposition

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during megasporogenesis such that callose began to accumulate abnormally from MEI

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and was deposited in large quantity at AME in the ovule of BL5+.

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ER stress and premature PCD induced in BL5+. Under stress conditions, the

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number of misfolded proteins will increase and may exceed the capacity of the ER

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quality-control system, thus inducing ER stress (Howell, 2013; Wan and Jiang, 2016).

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In BL5+, 78.95% genes in “RNA transcription”, 66.23% genes in “RNA processing”,

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57.69% genes in “protein synthesis initiation”, 58.33% genes in “protein synthesis

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elongation”, 82.46% genes in “ribosomal protein synthesis” and 60.91% genes in

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“ribosome biogenesis” were up-regulated at MMC (Supplemental Fig. S6), suggesting

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a greatly increased level of protein synthesis, which may increase the protein folding

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burden on ER. To detect whether ER stress occurred in BL5+ at MMC, we examined

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the expression of ER stress responsive genes in BL5+ and found that 10 such genes

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were significantly up-regulated including OsbZIP50, which is a key ER stress

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regulator in rice (Fig. 7A). The mRNA of OsbZIP50 was spliced when ER stress

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occurred (Hayashi et al., 2012). We then detected the unconventional splicing of

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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 www.plantphysiol.org on May 27, 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 www.plantphysiol.org on May 27, 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 www.plantphysiol.org on May 27, 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


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


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 www.plantphysiol.org on May 27, 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 www.plantphysiol.org on May 27, 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 www.plantphysiol.org on May 27, 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 www.plantphysiol.org on May 27, 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 www.plantphysiol.org on May 27, 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 www.plantphysiol.org on May 27, 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 www.plantphysiol.org on May 27, 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


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 www.plantphysiol.org on May 27, 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 www.plantphysiol.org on May 27, 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.

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