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. 2021 Apr 14;22(1):268.
doi: 10.1186/s12864-021-07503-7.

The molecular signatures of compatible and incompatible pollination in Arabidopsis

Chie Kodera  1   2 , Jérémy Just  3 , Martine Da Rocha  4 , Antoine Larrieu  3   5 , Lucie Riglet  3   6 , Jonathan Legrand  3 , Frédérique Rozier  3 , Thierry Gaude  3 , Isabelle Fobis-Loisy  7
Affiliations

The molecular signatures of compatible and incompatible pollination in Arabidopsis

Chie Kodera et al. BMC Genomics. .

Abstract

Background: Fertilization in flowering plants depends on the early contact and acceptance of pollen grains by the receptive papilla cells of the stigma. Deciphering the specific transcriptomic response of both pollen and stigmatic cells during their interaction constitutes an important challenge to better our understanding of this cell recognition event.

Results: Here we describe a transcriptomic analysis based on single nucleotide polymorphisms (SNPs) present in two Arabidopsis thaliana accessions, one used as female and the other as male. This strategy allowed us to distinguish 80% of transcripts according to their parental origins. We also developed a tool which predicts male/female specific expression for genes without SNP. We report an unanticipated transcriptional activity triggered in stigma upon incompatible pollination and show that following compatible interaction, components of the pattern-triggered immunity (PTI) pathway are induced on the female side.

Conclusions: Our work unveils the molecular signatures of compatible and incompatible pollinations both at the male and female side. We provide invaluable resource and tools to identify potential new molecular players involved in pollen-stigma interaction.

Keywords: Compatible / incompatible pollination; Male-female transcriptome; PTI pathway; Pollen-stigma interaction; RNA sequencing; SNPs.

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Conflict of interest statement

We have no competing interests.

Figures

Fig. 1
Fig. 1
Experimental and data analysis pipeline of SNP-based RNA-seq analysis. a Time course of sample collection. Flowers of Col-0/SRK14 were emasculated at stage 12, 16 h - 20 h before pollination with compatible (C24) or incompatible (C24/SCR14) pollen grains, respectively. Stigmas were harvested (dashed line) 0, 10, 60 min after compatible (C0, C10, C60) or incompatible (I10, I60) pollination for RNA extraction. Typical scanning electron microscope images observed 60 min after pollination, for compatible reaction (Col-0 /SRK14 x C24) with hydrated round pollen grains and pollen tubes, and incompatible reaction (Col-0 /SRK14 x C24/SCR14) with dehydrated ellipsoidal pollen grains and no pollen tube. Scale bar = 20 μm. b We performed whole genome sequencing and detected variants using GATK (McKenna et al., 2010). SNPs and short indels between Col-0/SRK14 and C24 were identified (1.). Then, we produced new reference genomes for Col-0/SRK14 and C24 introducing the identified SNPs into TAIR10 Col-0 genome (2.). After deriving predicted mRNA from the new genomes, we performed RNA-sequencing and got sex-specific isoform abundance by using a statistic tool ASE-TIGAR (Nariai et al., 2016) (3.). Read normalization and differentially expressed gene analysis were performed using DESeq2 (Love et al., 2014). Sequence quality was checked by FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc). DNA and RNA reads were cleaned with custom Perl scripts
Fig. 2
Fig. 2
Quality check of SNP-based RNA-seq analysis. a Estimated proportions of reads allocated to each tissue (31% pollen, 69% stigma) including reads without SNP (15%) that were equally distributed between pollen and stigma (shaded green, 7.5% + shaded purple, 7.5%). b Hexbin plot to visualize distribution of gene expression in stigma (nFPKM stigma) and pollen (nFPKM pollen) at C0. Gene count per hexagon is represented using a color gradient from light grey to orange. Genes without SNP are plotted outside the graph based on their stigma expression (nFPKM stigma)
Fig. 3
Fig. 3
Validation of the SNP-based analysis. a Heat map of Pearson’s correlation coefficient between the stigma / pollen transcripts from the SNP-based analysis and transcript information from previously published data. b Number of stigma or pollen (preferentially / specifically) -expressed genes at C0 selected by nFPKM (Additional file 3: Table S4). c RT-PCR and sequence analysis of stigma or pollen specifically-expressed genes at C0. Genes are selected among the top 20 specifically-expressed genes; their rank according to their expression level are presented. RNAs were extracted from pollinated stigmas at C0 and we analyzed their SNP-information by RT-PCR and sequencing. SNP number corresponds to the number of SNPs present in the sequenced regions. d Top ten GO term enrichment categories (biological processes) of stigma or pollen preferentially-expressed genes at C0. Enrichment analysis was performed with the one thousand top expressed genes in stigma (left) or pollen (right), respectively. Selection criteria for genes analysed in c (sex-specific), and d (sex-preferential), are described in text
Fig. 4
Fig. 4
Gene expression dynamics after pollination. a PCA of stigma (left) and pollen (right) transcripts. Biological replicates (dots or triangles) in all pollination conditions were analyzed. b Venn diagrams showing the number of upregulated (FC > 2) genes in stigma (left) and pollen (right), after compatible (green and violet) or incompatible (grey) pollinations. c Gene enrichment categories of up-regulated genes after incompatible pollination in stigma according to GO terms of biological processes. Genes exclusively induced after incompatible reaction (b, left; 4 + 104) were analyzed. Only enrichment categories with significant FDR (< 0.05) are shown. d Gene enrichment categories of up-regulated genes after compatible pollination in stigma according to GO terms of biological processes. Genes exclusively induced after compatible reaction (b, left; 158 + 163 + 623) were analyzed. Only top 20 enrichment categories are shown
Fig. 5
Fig. 5
PTI pathway predicted to be induced in stigma after KEGG pathway mapping analysis. PTI pathway modified according to the information from Bigeard et al. 2015, Bi et al. 2018, and Lian et al. 2018. Genes in green boxes were upregulated (FC > 2) after compatible pollination, at 10 min and 60 min (light green), or only at 60 min (dark green: genes with SNPs, hatched dark green: genes without SNP but predicted stigma specific with ECM, see Supplementary file 8 Table S10). Gene in grey box was upregulated (FC > 2) 60 min after incompatible pollination. White boxes mean genes without SNPs (with no ECM prediction), not induced, or induced after both (compatible/incompatible) pollination (see Supplementary file 7 Table S9). Genes in bold were expressed at C0; nFPKM (stigma) > 1. RLCKs = Receptor-Like Cytoplasmic Kinases
Fig. 6
Fig. 6
Selectively induced genes after pollination. From gene lists generated using a Venn diagram representation (Fig. 4b) we identified genes selectively induced 60 min after pollination in stigma (left) and in pollen (right) with criteria applied to the FC ratio between compatible and incompatible situations. The uppermost selectively induced genes sorted by FC ratio from the largest to the smallest (up to 10) are shown. * genes described in the discussion section
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