Genome-Wide Expression Profiling of the Arabidopsis Female Gametophyte Identifies Families of Small, Secreted Proteins
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ABSTRACT:
ORGANISM(S): Arabidopsis thaliana
PROVIDER: GSE8392 | GEO | 2007/10/10
SECONDARY ACCESSION(S): PRJNA101435
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 the 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.
2016-03-18 | PXD002215 | Pride
Project description:Polyadenylation of mRNAs is critical for their export from the nucleus, stability and efficient translation. The Arabidopsis thaliana genome encodes three isoforms of canonical nuclear poly(A) polymerase (PAPS) that redundantly polyadenylate the bulk of pre-mRNAs. However, their distinct mutant phenotypes and transcriptome studies have indicated that subsets of pre-mRNAs are preferentially polyadenylated by either PAPS1 or the two very similar PAPS2 and PAPS4 proteins. Such functional specialization raises the possibility of modulating the balance of activities between the isoforms to alter poly(A) lengths of sets of transcripts, providing an additional level of gene-expression control in plants. Here we test this notion by studying the function of PAPS1 in pollen-tube growth and guidance. Pollen tubes growing through female tissue acquire the competence to find ovules efficiently and upregulate PAPS1 expression more strongly than in vitro grown pollen tubes. Using the temperature-sensitive paps1-1 allele we show that PAPS1 activity during pollen-tube growth is required for full acquisition of competence, resulting in inefficient fertilization by paps1-1 mutant pollen tubes. While these mutant pollen tubes germinate and grow at the same rates as wild-type pollen tubes, they are compromised in locating the micropyles of ovules. Transcriptomic analyses indicate that previously identified competence-associated genes are less expressed in paps1-1 mutant than in wild-type pollen tubes. Estimating the poly(A)-tail lengths of transcripts in mutant and wild-type pollen tubes suggests that polyadenylation by PAPS1 is associated with reduced transcript abundance. Our results therefore suggest that PAPS1 upregulation during pollen-tube growth through the style plays a key role in the acquisition of competence and support the notion that plants can modulate the balance of activity between PAPS isoforms to regulate gene expression.
2023-02-21 | GSE207509 | GEO
Project description:Polyadenylation of mRNAs is critical for their export from the nucleus, stability and efficient translation. The Arabidopsis thaliana genome encodes three isoforms of canonical nuclear poly(A) polymerase (PAPS) that redundantly polyadenylate the bulk of pre-mRNAs. However, their distinct mutant phenotypes and transcriptome studies have indicated that subsets of pre-mRNAs are preferentially polyadenylated by either PAPS1 or the two very similar PAPS2 and PAPS4 proteins. Such functional specialization raises the possibility of modulating the balance of activities between the isoforms to alter poly(A) lengths of sets of transcripts, providing an additional level of gene-expression control in plants. Here we test this notion by studying the function of PAPS1 in pollen-tube growth and guidance. Pollen tubes growing through female tissue acquire the competence to find ovules efficiently and upregulate PAPS1 expression more strongly than in vitro grown pollen tubes. Using the temperature-sensitive paps1-1 allele we show that PAPS1 activity during pollen-tube growth is required for full acquisition of competence, resulting in inefficient fertilization by paps1-1 mutant pollen tubes. While these mutant pollen tubes germinate and grow at the same rates as wild-type pollen tubes, they are compromised in locating the micropyles of ovules. Transcriptomic analyses indicate that previously identified competence-associated genes are less expressed in paps1-1 mutant than in wild-type pollen tubes. Estimating the poly(A)-tail lengths of transcripts in mutant and wild-type pollen tubes suggests that polyadenylation by PAPS1 is associated with reduced transcript abundance. Our results therefore suggest that PAPS1 upregulation during pollen-tube growth through the style plays a key role in the acquisition of competence and support the notion that plants can modulate the balance of activity between PAPS isoforms to regulate gene expression.
2023-02-21 | GSE207285 | 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.
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.
2009-08-15 | E-GEOD-17343 | biostudies-arrayexpress
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.
2020-05-30 | GSE151462 | GEO
Project description:The female gametophyte of flowering plants, the embryo sac, develops within the diploid (sporophytic) tissue of the ovule. While embryo sac-expressed genes are known to be required at multiple stages of the fertilization process, the set of embryo sac-expressed genes has remained poorly defined. In particular, the set of genes responsible for mediating intracellular communication between the embryo sac and the male gametophyte, the pollen grain, is unknown. We used high-throughput cDNA sequencing and whole-genome tiling arrays to compare gene expression in wild-type ovules to that in dif1 ovules, which entirely lack embryo sacs, and myb98 ovules, which are impaired in pollen tube attraction. We identified nearly 400 genes that are downregulated in dif1 ovules. Seventy-eight percent of these embryo sac-dependent genes were predicted to encode for secreted proteins, and 60% belonged to multigenic families. Our results define a large number of candidate extracellular signaling molecules that may act during embryo sac development or fertilization; less than half of these are represented on the widely used ATH1 expression array. In particular, we found that 37 out of 40 genes encoding Domain of Unknown Function 784 (DUF784) domains require the synergid-specific transcription factor MYB98 for expression. Several DUF784 genes were transcribed in synergid cells of the embryo sac, implicating the DUF784 gene family in mediating late stages of embryo sac development or interactions with pollen tubes. The coexpression of highly similar proteins suggests a high degree of functional redundancy among embryo sac genes.
| S-EPMC2014789 | biostudies-literature
Project description:We perform a quantitative RNA-seq analysis of embryo sacs, comparator ovules with the embryo sacs removed, mature pollen, and seedlings to assist the identification of gametophyte functions in maize. Expression levels were determined for annotated genes in both gametophytes, and novel transcripts were identified from de novo assembly of RNA-seq reads. RNA-seq was performed on four tissue types: nine-day old, above-ground seedling (S); mature pollen (MP); embryo-sac-enriched samples with some remaining nucellar cells (ES); and ovules with embryo sacs removed (Ov).
2011-07-09 | E-MTAB-4128 | biostudies-arrayexpress
Project description:Flowering plants have immotile sperm that develop within pollen and must be carried to female gametes by a pollen tube. The pollen tube engages in molecular interactions with several cell types within the pistil and these interactions are essential for successful fertilization. We identified a group of three closely related pollen tube-expressed MYB transcription factors (MYB97, MYB101, MYB120), which are required for proper interaction of the pollen tube with the female gametophyte. These transcription factors are transcriptionally induced during growth in the pistil. They regulate a transcriptional network leading to proper differentiation and maturation of the pollen tube, promoting proper pollen tube-ovule interactions resulting in sperm release and double fertilization. We used microarrays to discover genes regulated by the transcription factors MYB97, MYB101 and MYB120 in pollen tubes growing through the pistil at 8 hours after pollination. Pistils were collected from ms1 (Male Sterile 1) pistils that were unpollinated, or pollinated with either wild type (Col-0) pollen or myb triple mutant (myb97-1, myb101-4, myb120-3) pollen for 8 hours. We sought to examine transcriptional changes that were taking place in pollen tubes before they reached ovules in wild type pollen tubes, and what portion of this transcriptional regulation was due to MYB97, MYB101 and MYB120. Analysis of growth in the pistil allows discovery of transcriptional changes taking place during pollen tube growth in its native environment, as opposed to mature pollen or in vitro grown pollen, which are essentially naive conditions, as neither have interacted with the pistil environment and any signalling factors found therein.
2013-06-18 | E-GEOD-44405 | biostudies-arrayexpress
Project description:The coordination of pollen tube (PT) growth, guidance and timely growth arrest and rupture mediated by PT-pistil interaction is crucial for the PT to transport sperm cells into ovules for double fertilization. The plasma membrane (PM) represents an important interface for cell–cell interaction, and PM proteins of PTs are pioneers for mediating PT integrity and interaction with pistils. Thus, understanding the mechanisms underlying these events is important for proteomics. Using the efficient aqueous polymer two-phase system and alkali buffer treatment, we prepared high-purity PM from mature and germinated pollen of rice. We used iTRAQ quantitative proteomic methods and identified 1,121 PM-related proteins (PMrPs) (matched to 899 loci); 192 showed differential expression in the two pollen cell types, 119 up- and 73 down-regulated during germination. The PMrP and differentially expressed PMrP sets all showed a functional skew toward signal transduction, transporters, wall remodeling/metabolism and membrane trafficking. Their genomic loci had strong chromosome bias. We found 37 receptor-like kinases (RLKs) from 8 kinase subfamilies and 209 transporters involved in flux of diversified ions and metabolites. In combination with the rice pollen transcriptome data, we revealed that in general, the protein expression of these PMrPs disagreed with their mRNA expression, with inconsistent mRNA expression for 74% of differentially expressed PMrPs. This study, for the first time, identified genome-wide pollen PMrPs, and provided insights into the membrane profile of receptor-like kinases and transporters important for pollen tube growth and interaction with pistils. These pollen PMrPs and their mRNAs showed discordant expression. This work provides novel resource and knowledge to further dissect mechanisms by which pollen or the PT controls PMrP abundance and monitors interactions and ion and metabolite exchanges with female cells in rice.
2017-01-16 | PXD004994 | Pride