Processes Underlying a Reproductive Barrier in indica- japonica Rice Hybrids Revealed by Transcriptome Analysis

Abstract

In rice (Oryza sativa), hybrids between indica and japonica subspecies are usually highly sterile, which provides a model system for 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, we performed transcriptomic analyses of pistils from rice variety Balilla (BL), Balilla with transformed ORF5+ (BL5+) producing sterile female gametes, and Balilla with transformed ORF3+ and ORF5+ (BL3+5+) producing fertile gametes. RNA sequencing of tissues collected before (MMC), during (MEI), and after (AME) meiosis of the megaspore mother cell detected 19,269 to 20,928 genes as expressed. Comparison between BL5+ and BL showed that ORF5+ induced differential expression of 8,339, 6,278, and 530 genes at MMC, MEI, and AME, respectively. At MMC, large-scale differential expression of cell wall-modifying genes and biotic and abiotic response genes indicated that cell wall integrity damage induced severe biotic and abiotic stresses. The processes continued to MEI and induced endoplasmic reticulum (ER) stress as indicated by differential expression of ER stress-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. Large numbers of cell wall modification and biotic and abiotic response genes were also induced at MMC but largely suppressed at MEI without inducing ER stress and programed cell death , producing fertile gametes. These results have general implications for the understanding of biological processes underlying reproductive barriers.

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, The MMC 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 μm. The gray bars at the top indicate the corresponding stage in transcriptome data.

Figure 2.

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

Figure 3.

Partial significantly (FDR < 0.005) enriched GO terms for up-regulated genes in BL5+ compared to BL. The color in each cell represents the significance of enrichment based on the FDR value. The cells without color indicate not significantly enriched. The full list of GO terms is given in Supplemental Table S3.

Figure 4.

Differential regulation of cell wall genes in BL5+ and BL3+5+ compared with BL. A, The number of differentially expressed 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 wall modification and degradation. The color in each cell represents the level of expression change based on the log₂ fold. The cell without color indicates the gene was not differentially expressed. Full list of genes and their expressions is given in Supplemental Table S5. C, Expression change of cellulose biosynthetic genes.

Figure 5.

Cellulose deposition in the mature ovules of BL, BL5+, and BL3+5+. A to F, Cellulose deposition 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 or blue fluorescence indicates where the cellulose accumulated. Bars = 50 μm. G, Cellulose content in the panicles of BL and BL5+. The data are means ± se (n = 3). ***Significant difference at P < 0.001.

Figure 6.

Callose deposition (green fluorescence) in the developing ovule of BL, BL5+, and BL3+5+ by aniline blue staining. A to F, The dyad stage. The red arrowheads indicate the callose bands around the dyad. G to L, The tetrad stage. The red arrowheads indicate the callose bands around the tetrad. M to R, The functional megaspore stage. The red arrowheads indicate the degenerated megaspores. S to X, The mature embryo-sac. I, O, and U, The gray arrowheads indicate abnormally deposited callose. Bars = 25 μm.

Figure 7.

Differential regulation of ER stress and PCD genes. A, Log₂ fold change of the expression level of ER stress and PCD genes in BL5+ against BL. The color in each cell represents the levels of expression change based on the log₂ fold. The cell without color indicates the gene was not differentially expressed. B, Splicing of OsbZIP50 mRNA 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 axis represents the expression level relative to profilin (Os06g05880). The data are means ± se (n = 3).

Figure 8.

Analysis of genes differentially expressed in BL3+5+ compared to BL. A, Number of DEGs detected in the BL3+5+/BL and 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” by MapMan at MMC, MEI, and AME, respectively. C, Partial significantly (FDR < 0.005) enriched GO terms for up-regulated genes in BL3+5+ compared with BL. The color in each cell represents the significance of enrichment based on the FDR value. The cells without color indicate not significantly enriched. The full list of GO terms is given in Supplemental Table S4.

Figure 9.

A model for S5 locus-mediated ovule abortion.