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PRP8A and PRP8B spliceosome subunits act coordinately to control pollen tube attraction in Arabidopsis thaliana

ABSTRACT: Purpose: Alternative splicing is fundamental for post-transcriptional regulation and proteome diversity. The goals of this study are to compare transcriptome and splicing profiling (RNA-seq) between wild type and prp8a prp8b mutant ovules of the spliceosome subunit and define the molecular signature of prp8a prp8b pollen tube attraction phenotype. Methods: mRNA profiles from mature ovules of 6-weeks-old wild-type (WT) and pre-mRNA processing factor 8 (PRP8Aa prp8bb) Arabidopsis plants were generated by deep sequencing, in triplicates, using Illumina HiSeq4000 100bp paired-end reads. The sequence reads that passed quality filters were were mapped to TAIR10 whole genome and analyzed for differential expression, differential exone usage and intron retention as indicated in DATA PROCESSING PIPELINE section. Results: Using an optimized data analysis workflow, about 15 million sequence read pairs per sample were mapped to the Arabidopsis genome (TAIR10). Approximately 2.9% of the transcripts showed differential expression between the WT and PRP8Aa prp8bb ovules, with a fold change ≥1.5 and p value <0.05. Analysis for differential gene expression, exon usage and intron retention with DESeq2 v1.22.1 and DEXseq v1.28.0 or IRFinder v1.2.3 respectively, uncovered several as yet uncharacterized genes that may contribute to pollen tube attraction and female gametophyte cell fate specification. Conclusions: Our work has uncovered a molecular signature through which PRP8A/PRP8B subunits act redundantly to define male-female signaling competence for successful pollen tube attraction in Arabidopsis. Application of DESeq2 algorithms to our ovule RNA-seq data identified downregulation of over 50 different CRP genes with yet unknown function from the synergid and the central cells including all LURE pollen tube attractants. Whereas use of DEXseq workflow, revealed mis-splicing of key genes involved embryo sac specificiation and genes of the secretory pathway. We concluded that 100bp paired-end RNAseq was a sufficient compromise for detection of differential gene expression and splice isoforms, however, our experiment would have benefited with more number of replicates.

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE151462 | GEO | 2020/05/30

REPOSITORIES: GEO

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Project description:As in animals, cell-cell communication plays pivotal role in male-female recognition during plant sexual reproduction. Prelaid peptides secreted from the female reproductive tissues guide pollen tubes towards ovules for fertilization. However, the elaborate mechanisms for this dialogue have remained elusive, particularly from the male perspective. We perform genome-wide quantitative liquid chromatography coupled tandem mass spectrometry of a pistil-stimulated pollen tube secretome and identify 801 pollen tube-secreted proteins. Interestingly, in silico analysis reveals that the pollen tube-secretome is dominated by unconventional-type secreted proteins representing 57% of the total secretome. In support, we show that unconventional-type protein, translationally controlled tumor protein, is secreted to the apoplast. Remarkably, we discover that this protein could be secreted by infiltrating through the initial phases of the conventional secretory pathway and could reach the apoplast via exosomes as demonstrated by co-localization with Oleisin1 exosome marker. We demonstrate that Arabidopsis thaliana translationally controlled tumor protein-knockdown plants have pollen tubes that poorly navigate to the target ovule, and theÂ knocked down allele is poorly transmitted through the male. We show that regulators of the endoplasmic reticulum-trans-golgi network protein secretory pathway control secretion of pollen tube-secreted cysteine-rich proteins, including pollen tube attractants, and are essential for pollen tube growth and guidance, as well as ovule-targeting competence. This work, the first pollen tube secretome study, identifies novel genome-wide pollen tube-secreted proteins with potential function in pollen tube-ovule guidance for sexual reproduction. Functional analysis highlights a potential mechanism for pollen tube unconventional protein secretion and reveals likely regulators of pollen tube protein secretion. The association of pollen tube-secreted proteins with marker proteins shown to be secreted via exosomes in other species suggest secretion via exosomes as a possible mechanism for cell-cell communication between the pollen tube and female reproductive cells. For processed dataset with quantitative information, see Hafidh S, Potesil D, Fila J et al. Genome Bilogy 2016.

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PRP8A and PRP8B spliceosome subunits act coordinately to control pollen tube attraction in Arabidopsis thaliana

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