BACKGROUND: As in animals, cell-cell communication plays a 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. RESULTS: We performed genome-wide quantitative liquid chromatography-tandem mass spectrometry analysis of a pistil-stimulated pollen tube secretome and identified 801 pollen tube-secreted proteins. Interestingly, in silico analysis reveals that the pollen tube secretome is dominated by proteins that are secreted unconventionally, representing 57 % of the total secretome. In support, we show that an unconventionally secreted protein, translationally controlled tumor protein, is secreted to the apoplast. Remarkably, we discovered 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 translationally controlled tumor protein-knockdown Arabidopsis thaliana plants produce pollen tubes that navigate poorly to the target ovule and that the mutant allele is poorly transmitted through the male. Further, we show that regulators of the endoplasmic reticulum-trans-Golgi network protein secretory pathway control secretion of Nicotiana tabacum Pollen tube-secreted cysteine-rich protein 2 and Lorelei-like GPI-anchor protein 3 and that a regulator of endoplasmic reticulum-trans-Golgi protein translocation is essential for pollen tube growth, pollen tube guidance and ovule-targeting competence. CONCLUSIONS: This work, the first study on the pollen tube secretome, identifies novel genome-wide pollen tube-secreted proteins with potential functions in pollen tube guidance towards ovules for sexual reproduction. Functional analysis highlights a potential mechanism for unconventional secretion of pollen tube proteins and reveals likely regulators of conventional pollen tube protein secretion. The association of pollen tube-secreted proteins with marker proteins shown to be secreted via exosomes in other species suggests exosome secretion is a possible mechanism for cell-cell communication between the pollen tube and female reproductive cells.
- MeSH
- Arabidopsis genetika fyziologie MeSH
- fertilizace * MeSH
- opylení MeSH
- proteom * MeSH
- pylová láčka genetika růst a vývoj metabolismus MeSH
- rostlinné proteiny genetika metabolismus MeSH
- sekreční dráha * MeSH
- tabák genetika fyziologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Tobacco mature pollen has extremely desiccated cytoplasm, and is metabolically quiescent. Upon re-hydration it becomes metabolically active and that results in later emergence of rapidly growing pollen tube. These changes in cytoplasm hydration and metabolic activity are accompanied by protein phosphorylation. In this study, we subjected mature pollen, 5-min-activated pollen, and 30-min-activated pollen to TCA/acetone protein extraction, trypsin digestion and phosphopeptide enrichment by titanium dioxide. The enriched fraction was subjected to nLC-MS/MS. We identified 471 phosphopeptides that carried 432 phosphorylation sites, position of which was exactly matched by mass spectrometry. These 471 phosphopeptides were assigned to 301 phosphoproteins, because some proteins carried more phosphorylation sites. Of the 13 functional groups, the majority of proteins were put into these categories: transcription, protein synthesis, protein destination and storage, and signal transduction. Many proteins were of unknown function, reflecting the fact that male gametophyte contains many specific proteins that have not been fully functionally annotated. The quantitative data highlighted the dynamics of protein phosphorylation during pollen activation; the identified phosphopeptides were divided into seven groups based on the regulatory trends. The major group comprised mature pollen-specific phosphopeptides that were dephosphorylated during pollen activation. Several phosphopeptides representing the same phosphoprotein had different regulation, which pinpointed the complexity of protein phosphorylation and its clear functional context. Collectively, we showed the first phosphoproteomics data on activated pollen where the position of phosphorylation sites was clearly demonstrated and regulatory kinetics was resolved.
- MeSH
- fosfoproteiny chemie metabolismus MeSH
- kinetika MeSH
- proteomika metody MeSH
- pyl metabolismus MeSH
- regulace genové exprese u rostlin MeSH
- rostlinné proteiny chemie metabolismus MeSH
- tabák genetika metabolismus MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- vazebná místa MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The journey undertaken by the pollen tube in angiosperms to reach the deeply embedded female gametophyte for fertilization involves persistent guidance by the female gametophyte and accurate perception of the signals by the pollen tube. Several ovule-secreted peptides have been identified. Nevertheless, there are no exact findings on how these signals are perceived by the pollen tube. As a novel approach, we have improvised a modified SIV (semi-in vivo) technique, SIV-PS (SIV pollen tube secretome), to perform gel-free LC-MS/MS for high-throughput analysis of pollen-tube-secreted proteins. Our approach has led to the identification of over 1400 protein groups. Among them are pollen-tube-secreted ligands and receptor proteins representing potential male components in perceiving ovule-emitted cues for guidance. The present article reviews the missing link in pollen tube perception and showcases the improvised SIV-PS as a tool for high-throughput and targeted study of the pollen tube secretome.
Mature pollen represents an extremely resistant quiescent structure surrounded by a tough cell wall. After its hydration on stigma papillary cells, pollen tube growth starts rapidly. Massive metabolic changes are likely to be accompanied by changes in protein phosphorylation. Protein phosphorylation belongs among the most rapid post-translational modifications. To date, only Arabidopsis thaliana and tobacco (Nicotiana tabacum) mature pollen have been subjected to phosphoproteomic studies in order to identify the phosphoproteins present. In the present mini-review, Arabidopsis and tobacco datasets were compared with each other. The representation of the O-phosphorylated amino acids was compared between these two datasets, and the putative pollen-specific or pollen-abundant phosphopeptides were highlighted. Finally, the phosphorylation sites common for both Arabidopsis and tobacco phosphoproteins are listed as well as the phosphorylation motifs identified.
The transition between the quiescent mature and the metabolically active germinating pollen grain most probably involves changes in protein phosphorylation status, since phosphorylation has been implicated in the regulation of many cellular processes. Given that, only a minor proportion of cellular proteins are phosphorylated at any one time, and that phosphorylated and nonphosphorylated forms of many proteins can co-exist within a cell, the identification of phosphoproteins requires some prior enrichment from a crude protein extract. Here, we have used metal oxide/hydroxide affinity chromatography (MOAC) based on an aluminum hydroxide matrix for this purpose, and have generated a population of phosphoprotein candidates from both mature and in vitro activated tobacco pollen grains. Both electrophoretic and nonelectrophoretic methods, allied to MS, were applied to these extracts to identify a set of 139 phosphoprotein candidates. In vitro phosphorylation was also used to validate the spectrum of phosphoprotein candidates obtained by the MOAC phosphoprotein enrichment. Since only one phosphorylation site was detected by the above approach, titanium dioxide phosphopeptide enrichment of trypsinized mature pollen crude extract was performed as well. It resulted in a detection of additional 51 phosphorylation sites giving a total of 52 identified phosphosites in this set of 139 phosphoprotein candidates.
- MeSH
- 2D gelová elektroforéza MeSH
- chromatografie afinitní metody MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- fosfoproteiny analýza chemie izolace a purifikace MeSH
- fosforylace MeSH
- molekulární sekvence - údaje MeSH
- proteom analýza chemie MeSH
- pyl chemie MeSH
- rostlinné proteiny analýza chemie MeSH
- sekvence aminokyselin MeSH
- sekvenční seřazení MeSH
- tabák chemie MeSH
- titan MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Many flowering plants produce bicellular pollen. The two cells of the pollen grain are destined for separate fates in the male gametophyte, which provides a unique opportunity to study genetic interactions that govern guided single-cell polar expansion of the growing pollen tube and the coordinated control of germ cell division and sperm cell fate specification. We applied the Agilent 44 K tobacco gene chip to conduct the first transcriptomic analysis of the tobacco male gametophyte. In addition, we performed a comparative study of the Arabidopsis root-hair trichoblast transcriptome to evaluate genetic factors and common pathways involved in polarized cell-tip expansion. RESULTS: Progression of pollen grains from freshly dehisced anthers to pollen tubes 4 h after germination is accompanied with > 5,161 (14.9%) gametophyte-specific expressed probes active in at least one of the developmental stages. In contrast, > 18,821 (54.4%) probes were preferentially expressed in the sporophyte. Our comparative approach identified a subset of 104 pollen tube-expressed genes that overlap with root-hair trichoblasts. Reverse genetic analysis of selected candidates demonstrated that Cu/Zn superoxide dismutase 1 (CSD1), a WD-40 containing protein (BP130384), and Replication factor C1 (NtRFC1) are among the central regulators of pollen-tube tip growth. Extension of our analysis beyond the second haploid mitosis enabled identification of an opposing-dynamic accumulation of core regulators of cell proliferation and cell fate determinants in accordance with the progression of the germ cell cycle. CONCLUSIONS: The current study provides a foundation to isolate conserved regulators of cell tip expansion and those that are unique for pollen tube growth to the female gametophyte. A transcriptomic data set is presented as a benchmark for future functional studies using developing pollen as a model. Our results demonstrated previously unknown functions of certain genes in pollen-tube tip growth. In addition, we highlighted the molecular dynamics of core cell-cycle regulators in the male gametophyte and postulated the first genetic model to account for the differential timing of spermatogenesis among angiosperms and its coordination with female gametogenesis.
- MeSH
- Arabidopsis genetika MeSH
- buněčný cyklus genetika MeSH
- gametogeneze rostlin MeSH
- genový knockdown MeSH
- klíčení MeSH
- kořeny rostlin genetika MeSH
- pyl genetika MeSH
- pylová láčka růst a vývoj MeSH
- regulace genové exprese u rostlin MeSH
- RNA rostlin genetika MeSH
- sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů MeSH
- tabák genetika MeSH
- transkriptom MeSH
- vývojová regulace genové exprese MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
The mRNA-protein complexes (mRNPs, Messenger ribonucleoprotein particles) are the "couriers" of the modern eukaryotes that process, store and deliver messages (transcripts) from the nucleus to the appropriate subcellular compartments and beyond. Presence of mRNPs arbitrates the posttranscriptional control of gene expression by editing the precursor RNA to maturity, postulate its subcellular localization and/or storage and dictate its fate once in the cytoplasm; either to be translated or dispensed through mRNA degradation. Initiation of transcription is coupled with processing of the transcribed message and the immediate association of the transcript with a set of structural and regulatory proteins. Per se, mRNP complexes sub-optimize transcription by recruiting RNA-binding proteins which are the core component of the RNP activities that culminate overall distribution and abundance of individual proteins. This asymmetric distribution of the mRNA is the determinant of protein gradient and is known to influence cell polarity, cell fate and overall patterning during development. Embryo patterning in Drosophila, polarization of maternal mRNA to daughter cell in budding yeast and directional growth of mammalian neural cell and pollen tubes of flowering plants, are the most prominent examples of mRNP facilitated posttranscriptional control, influencing cell fates and patterns of development.This chapter addresses the current knowledge on the mechanisms of posttranscriptional control reinforced by the formation of RNP particles and reviews differences in the underlying mechanisms. The outline of the chapter encompasses step-wise cellular processes leading to the formation of mRNPs and its implication to cellular activities. A dedicated section is also integrated discussing the recent findings on the unique mechanism of RNP formation in the male gametophyte of Nicotiana tabaccum. A proposed model outlines the network of posttranscriptional control with a focus on the role of RNPs is also presented aiming to stimulate future research with a perspective of advancing our knowledge on the subject and its plausible application in improving food quality.
- MeSH
- biologické modely MeSH
- cytoskelet metabolismus MeSH
- lidé MeSH
- messenger RNA genetika metabolismus MeSH
- posttranskripční úpravy RNA MeSH
- proteiny vázající RNA genetika metabolismus MeSH
- ribonukleoproteiny genetika metabolismus MeSH
- stabilita RNA MeSH
- transport proteinů MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Turistické slovníky
438 s. : il.
- Klíčová slova
- francouzština, slovníky kapesní,
- Publikační typ
- slovníky MeSH
- NLK Obory
- lingvistika, lékařská terminologie
