Pollen Aerobiome
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA848427 | ENA |
REPOSITORIES: ENA
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Project description: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.
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Project description:Pollen tubes extend through pistil tissues and are guided to ovules where they release sperm for fertilization. Although pollen tubes can germinate and elongate in a synthetic medium, their trajectory is random and their growth rates are slower compared to growth in pistil tissues. Furthermore, interaction with the pistil renders pollen tubes competent to respond to guidance cues secreted by specialized cells within the ovule. The molecular basis for this potentiation of the pollen tube by the pistil remains uncharacterized. We used a surgical procedure to obtain large quantities of uncontaminated pollen tubes that grew through the pistil and defined their transcriptome by microarray analysis. We also characterized the transcriptome of in vitro-grown pollen tubes (for 0.5hours or 4hours) and dessicated mature pollen in Arabidopsis.
2009-07-28 | GSE17343 | GEO
Project description:Pollen tubes extend through pistil tissues and are guided to ovules where they release sperm for fertilization. Although pollen tubes can germinate and elongate in a synthetic medium, their trajectory is random and their growth rates are slower compared to growth in pistil tissues. Furthermore, interaction with the pistil renders pollen tubes competent to respond to guidance cues secreted by specialized cells within the ovule. The molecular basis for this potentiation of the pollen tube by the pistil remains uncharacterized. We used a surgical procedure to obtain large quantities of uncontaminated pollen tubes that grew through the pistil and defined their transcriptome by microarray analysis. We also characterized the transcriptome of in vitro-grown pollen tubes (for 0.5hours or 4hours) and dessicated mature pollen in Arabidopsis. Experiment Overall Design: Pollen and pollen tubes were collected as described in the protocols section for RNA extraction and hybridization on Affymetrix ATH1 Genechip microarrays.
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