The Molecular Dialog between Flowering Plant Reproductive Partners Defined by SNP-Informed RNA-Sequencing

Abstract

The molecular interactions between reproductive cells are critical for determining whether sexual reproduction between individuals results in fertilization and can result in barriers to interspecific hybridization. However, it is a challenge to define the complete molecular exchange between reproductive partners because parents contribute to a complex mixture of cells during reproduction. We unambiguously defined male- and female-specific patterns of gene expression during Arabidopsis thaliana reproduction using single nucleotide polymorphism-informed RNA-sequencing analysis. Importantly, we defined the repertoire of pollen tube-secreted proteins controlled by a group of MYB transcription factors that are required for sperm release from the pollen tube to the female gametes, a critical barrier to interspecific hybridization. Our work defines the pollen tube gene products that respond to the pistil and are required for reproductive success; moreover, we find that these genes are highly evolutionarily plastic both at the level of coding sequence and expression across A. thaliana accessions.

Figure 1.

Pollen- and Pistil-Specific Gene Expression Can Be Distinguished in Pollinated Pistils Using SNPs in RNA-Seq Reads. (A) Experimental samples prepared for RNA-seq. (B) The distribution of SNPs across indicated gene regions in our Cvi versus Col SNP database (percentage is given above bars; black represents transcribed regions; gray represents untranscribed regions of transposable elements [TE]). (C) Reads from three biological replicates of a pollinated pistil RNA-seq experiment were analyzed to determine the number with Col or Cvi SNPs; the numbers of total and sex-specific reads are given. Venn diagram is not proportional to number of included elements. (D) and (E) Scatterplots of mean reads/gene (three biological replicates). (D) Pistil reads (y axis) are plotted against pollen reads (x axis) for all genes with SNPs detected in pollinated pistils. The y axis is 10-fold greater than the x axis. (E) Total reads/gene are plotted for unpollinated pistils (y axis) and pollinated pistils (x axis). All genes are plotted as colored dots indicating sex-specific expression: blue, pollen specific; red, pistil specific, gray, genes without SNPs. (F) Quantification of genes expressed in pollen, pistil, or both tissues. Supporting bioinformatic analysis is shown in Supplemental Figures 1, 3, and 4.

Figure 2.

SNP-Informed RNA-Seq Defines the Male and Female Contributions to Gene Expression during Reproduction. (A) to (F) Plots of reads over gene models at representative loci. Coverage (y axis) = log₁₀ number of reads counted over each nucleotide. Gray color represents reads without SNPs. In regions with SNPs (green line), reads are divided into sex-specific components: red = reads with the Cvi SNP (pistil parent) and blue = reads with the Col SNP (pollen parent). (A) to (C) Pistil-expressed genes. (D) to (F) Pollen-expressed genes. (G) to (J) Genes that are abundant in pollinated pistils and are expressed in both pollen and pistil. Exons are represented by black bars, introns by black lines, and polarity of transcription by arrowheads. (K) Pearson’s R correlation values are graphed with the indicated scale comparing pollen tube- and pistil-expressed genes defined by SNP-informed RNA sequencing of pollinated pistils with previously published microarray experiments (references given in text). Only genes with one unambiguous microarray ID were analyzed. Supporting bioinformatic analysis is shown in Supplemental Figure 5.

Figure 3.

SNP-Informed RNA-Seq Defines MYB-Responsive Genes in the Pollen Tube and Pistil. (A) Experimental samples prepared for RNA-seq. (B) and (C) Scatterplots of all average reads (ignoring SNPs) in wild type versus myb triple-mutant pollinations. (B) Differentially expressed genes identified before division of reads based upon SNPs are indicated (purple, 318 genes, adjusted P value < 0.05). (C) The color code of the scatterplot in (B) was altered to indicate genes with significant changes in the pollen (reads with Col SNP, light blue, n = 278, P value < 0.05, DESeq2) or pistil (reads with Cvi SNP, pink, n = 34, adjusted P value < 0.05) components of their expression profile. (D) A comparison of the differentially expressed genes identified with or without SNP analysis (P value < 0.05, DESeq2). (E) Differential expression analysis was performed again comparing genes identified after analysis of reads without SNPs with those identified based on differential abundance (P value < 0.05, DESeq2) of pollen (Col) or pistil (Cvi) reads. Differentially expressed genes identified by both analyses are highlighted in dark colors, and genes only identified by SNP-informed RNA-seq are highlighted in light colors (blue, pollen reads; red, pistil reads). The total number of differentially expressed genes is shown next to each category, along with the number that were downregulated (down) and upregulated (up) in mutant compared with wild-type pollinations. (F) Representative read coverage graphs (as in Figure 2) of MYB-dependent genes (AT1G04310 and AT1G33470). Top row, Cvi x Col-0 (wild-type) pollinations; bottom row, Cvi x myb triple-mutant pollinations. Supporting bioinformatic analysis is shown in Supplemental Figure 2. Supporting bioinformatic analysis is shown in Supplemental Figure 6.

Figure 4.

Pollen-Specific Gene Expression of Three MYB-Dependent Genes Detected by SNP-Informed RNA-Seq. (A), (H), and (O) Sequencing reads over SNPs identifying potential MYB target genes as pollen specific (as in Figure 2). (B) to (F) SUC9:SUC9:GFP (AT5G06170) and LAT52:DsRed. (I) to (M) PL or PL:PL:GFP (AT5G09280) and LAT52:DsRed. (P) to (T) ACA4:ACA4:GFP (AT4G20990) and LAT52:DsRed. (B), (I), and (P) Representative SIV pollen tube growth experiments on ms1 pistil explants (epifluorescent micrographs). In each, the left image is the red channel (LAT52:DsRed) signal, which is present in the pollen grain and the tube. The right image is the green channel (GFP) signal. The autofluorescence of the ms1 stigma explant is visible in each panel. Bars = 500 µm. (C), (J), and (Q) Confocal micrographs of SIV-grown pollen tubes: left panel, GFP; middle panel, DsRED; right panel, merge. Bars = 20 µm. (D), (K), and (R) Representative confocal optical sections of unpollinated pistils; the merge of differential interference contrast and GFP channels is shown. Arrows indicate expression of the GFP fusion protein in the female gametophyte. Bars = 100 µm. (E), (L), and (S) Close up of unpollinated ovules from (D), (K), and (R). Bars = 100 µm, projection of red and green channels. (F), (M), and (T) Projections of confocal micrographs of pollinated pistils. Red and green channels are merged, and arrows indicate regions of strong coexpression of DsRed and the indicated GFP fusion protein (yellow) in the style. Twelve hours after pollination. Bars = 100 µm. (G), (N), and (U) Close-up of ovules targeted by GFP reporter constructs from (F), (M), and (T). Bars = 100 µm. Supporting confocal micrographs are shown in Supplemental Figure 7. Supporting RT-qPCR analysis is presented in Supplemental Figure 8.

Figure 5.

Reproductive Gene Regulation Is Highly Divergent between Col and Cvi. (A) Hierarchical clustering of all RNA-seq samples identifies accession-specific clusters (DESeq2). (B) Intersection of all expressed genes (mean expression >100 reads) from self and inter-accession crosses. Venn diagram is not proportional to number of included elements. (C) Differential gene expression observed between Col and Cvi 8 h after self-pollination. Differentially expressed genes are colored purple (adjusted P value < 0.005, DESeq2). (D) to (F) Heat maps of Col-specific gene expression; colors represent read volume, with scales below indicating the number of reads for color value. (D) Defensin-like genes. (E) Low-molecular-weight cysteine-rich genes. (F) Toll-interleukin receptor-nucleotide binding site-leucine rich repeat (TIR-NBS-LRR) type disease resistance proteins. (G) Differential gene expression between self-pollinated Col and Cvi pistils. A total of 2877 differentially expressed were identified following pollination; 1640 transcripts were higher in inter-accession (Cvi x Col) pollinations, and 1238 transcripts were higher in self-pollinations (Cvi x Cvi). Adjusted P value < 0.005 (DESeq2). (H) Sex-specific transcript identity of (A). Red dots, Cvi-specific reads; blue dots, Col-specific reads; gray dots, no SNPs for assignment.

Figure 6.

MYB Transcription Factors Control Expression of Thionin CRPs (CRP2460). (A) A phylogenetic tree of the CRP2460 protein coding sequences (neighbor-joining, jukes-cantor). Two MYB-dependent members of other CRP groups (CRP2465 and CRP2530) are included for comparison. (B) Expression heat map of the CRP2460 transcripts in pistils. Cvi Unpoll, unpollinated Cvi pistils (collected 8 h after time when pollination would have been done); Cvi x Col, Cvi pistils pollinated with Col pollen for 8 h; Cvi x myb, Cvi pistils pollinated with myb triple-mutant pollen for 8 h; Col x Col, Col pistils pollinated with Col pollen for 8; Cvi x Cvi, Cvi pistils pollinated with Cvi pollen for 8 h. Sequencing data are shown in three columns per condition, with one column per biological replicate, and color intensity is based on read number (see scale). Asterisks indicate statistically significant gene expression differences between Cvi x Col and Cvi x myb triple-mutant pollinations (adjusted P value < 0.005, DESeq2). Green plus sign indicates the presence of SNPs in these CRP genes. Ka/Ks, numbers indicate Ka/Ks ratio between CRP and homolog in A. lyrata. Gray box indicates no homolog present for this CRP. (C) Representative sequencing data for two MYB-dependent CRP2460 genes, AT1G58242 and AT3G24465.

Figure 7.

Elevated Ka/Ks Rates in CRP Genes. (A) Percentage of genes in indicated gene families with homologs identified in given species identified by Lloyd et al. (2015). (B) Average Ka/Ks of all homologs found between A. thaliana and the indicated species. Bars are sd (sample sizes vary and are defined in [A]; Lloyd et al., 2015). (C) Percentage of homologous gene pairs out of the total number of family members with a Ka/Ks >1 (indicator of positive selection) for each respective species. The key pertains to (A) to (C).