Translatome of floral buds, in vivo pollinated pollen tubes, and in vitro cultured pollen grains/tubes from LAT52-HF-RPL18 Arabidopsis
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ABSTRACT: Little is known of the transcriptome of in vivo-grown pollen tubes, due to the difficulty of collection of pollen tubes elongating within the maternal gynoecium.We obtained the mRNAs undergoing translation (the translatome) of in vivo-grown pollen tubes from self-pollinated gynoecia of Arabidopsis thaliana(Col-0). Transgenic Arabidopsis plants (LAT52-HF-RPL18) harboring an epitope tagged ribosomal protein L18 driven by the pollen specific promoter (ProLAT52) were used for mRNA-ribosome complex isolation. After collection of polyribosomal (polysomal) complexes from self-pollinated (in vivo), unpollinated styles (buds), and in vitro-cultured pollen tubes, the actively translated mRNAs (the translatome) were purified, amplified to antisense RNA (aRNA). These aRNAs were hybridized to microarrays.Three independent biological replicates samples of aRNA from Bud, in vivo, and in vitro polysomal mRNA (translatomes) were hybridized to GeneChips to produce CEL files.
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.
Project description:Self-inhibition of pollen tubes plays a key role in SI, but the underlying mechanism in Camellia oleifera is poorly understood. Collection of secreted proteins from Camellia oleifera pollen tubes and ovaries for high-throughput sequencing.
Project description:Mitogen activated protein kinases (MAPKs) and phosphoinositides contribute to establishment and maintenance of polarity in eukaryotic cells. However, interactions between these important signaling pathways are largely unclear. Pollen tubes are an important model for the study of polarized cell expansion, in which PI4P 5-kinases control apical secretion and MAPKs contribute to pollen tube guidance. Here we report on the regulation of the Arabidopsis PI4P 5-kinase, AtPIP5K6, by the MAPK, MPK6. Purified recombinant AtPIP5K6 was phosphorylated in vitro upon incubation with pollen tube extracts. Non-targeted in-gel-kinase assays of pollen tube extracts followed by mass-spectrometry identified MPK6 as a candidate protein kinase upstream of AtPIP5K6. MPK6 interacted with AtPIP5K6 in yeast-two-hybrid tests, and bimolecular fluorescence complementation verified the interaction at the apical plasma membrane of pollen tubes. Recombinant AtPIP5K6 was phosphorylated in vitro by recombinant MPK6, resulting in reduced catalytic activity of AtPIP5K6. Alanine-substitution of phosphorylation sites, T590A and T597A, abrogated phosphorylation of AtPIP5K6 by MPK6 and prevented the in vitro inhibition. A T597D substitution resulted in reduced catalytic activity of the modified AtPIP5K6 protein in vitro. Coexpression of MPK6 with AtPIP5K6 in pollen tubes resulted in attenuation of PI4P 5-kinase-mediated polarity defects in vivo. Reciprocally, AtPIP5K6 effects were enhanced upon concomitant RNAi-inhibition of endogenous tobacco MAPKs. Both effects were absent when a AtPIP5K6 T590A T597A-variant was expressed, which could no longer be phosphorylated. We conclude that MPK6 limits PI4P 5-kinase-dependent polarized secretion to effect pollen tube guidance, revealing a new link between conserved signaling pathways with potential ramifications across eukaryotic kingdoms.
Project description:Little is known of the transcriptome of in vivo-grown pollen tubes, due to the difficulty of collection of pollen tubes elongating within the maternal gynoecium.We obtained the mRNAs undergoing translation (the translatome) of in vivo-grown pollen tubes from self-pollinated gynoecia of Arabidopsis thaliana(Col-0).
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.
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.
Project description:Arabinogalactan proteins are proteoglycans known to have important roles during cell growth and development, namelly during pollen tube growth. A microarray experiment was performed on agp6 agp11 pollen tubes to search for genetic interactions in the context of pollen tube growth. RNA from wt and mutant pollen tubes was extracted after 8h of in vitro germination and hybridized on Affimetrix microarrays.
Project description:Along with lipidomic and metabolomic analyses, we analysed the effect of short-term heat stress on Nicotiana tabacum pollen tubes. Tubes were either grown for 3 hours at room temperature, for 6 hours at room temperature or for 3 hours at room temperature and then 37 °C for another 3 hours.
Project description:Pollen germination and subsequent pollen tube elongation are essential for successful land plant reproduction. These processes are achieved through well-documented activation of membrane trafficking and cell metabolism. Despite this, our knowledge of the dynamics of cellular phospholipids remains scarce. In this project, we analyzed the turnover of the glycerolipid composition during the establishment of cell polarity and elongation processes in tobacco pollen and described the proteo-lipid composition of pollen plasma membrane-enriched. To achieve this, we have combined several techniques, such as lipidomics, live-cell microscopy, and plasma membrane isolation coupled with mass spectrometry-based proteomic characterization.
Project description:In this study, RNA-seq based comparative transcriptome analysis was used to study the genetic response of maize silk to pollen tube penetration and in comparison to the fungal invasion of Fusarium graminearum and Ustilago maydis. RNA-seq libraries of 8 tissues were generated from leaf, root, seed, pollen tube, silk, pollinated silk, infected silk with Fusarium and infected silk with Ustilago.