Angiosperm mature pollen represents a quiescent stage with a desiccated cytoplasm surrounded by a tough cell wall, which is resistant to the suboptimal environmental conditions and carries the genetic information in an intact stage to the female gametophyte. Post pollination, pollen grains are rehydrated, activated, and a rapid pollen tube growth starts, which is accompanied by a notable metabolic activity, synthesis of novel proteins, and a mutual communication with female reproductive tissues. Several angiosperm species (Arabidopsis thaliana, tobacco, maize, and kiwifruit) were subjected to phosphoproteomic studies of their male gametophyte developmental stages, mostly mature pollen grains. The aim of this review is to compare the available phosphoproteomic studies and to highlight the common phosphoproteins and regulatory trends in the studied species. Moreover, the pollen phosphoproteome was compared with root hair phosphoproteome to pinpoint the common proteins taking part in their tip growth, which share the same cellular mechanisms.
The nascent polypeptide-associated (NAC) complex was described in yeast as a heterodimer composed of two subunits, α and β, and was shown to bind to the nascent polypeptides newly emerging from the ribosomes. NAC function was widely described in yeast and several information are also available about its role in plants. The knock down of individual NAC subunit(s) led usually to a higher sensitivity to stress. In Arabidopsis thaliana genome, there are five genes encoding NACα subunit, and two genes encoding NACβ. Double homozygous mutant in both genes coding for NACβ was acquired, which showed a delayed development compared to the wild type, had abnormal number of flower organs, shorter siliques and greatly reduced seed set. Both NACβ genes were characterized in more detail-the phenotype of the double homozygous mutant was complemented by a functional NACβ copy. Then, both NACβ genes were localized to nuclei and cytoplasm and their promoters were active in many organs (leaves, cauline leaves, flowers, pollen grains, and siliques together with seeds). Since flowers were the most affected organs by nacβ mutation, the flower buds' transcriptome was identified by RNA sequencing, and their proteome by gel-free approach. The differential expression analyses of transcriptomic and proteomic datasets suggest the involvement of NACβ subunits in stress responses, male gametophyte development, and photosynthesis.
- MeSH
- alely MeSH
- Arabidopsis fyziologie MeSH
- fenotyp MeSH
- geneticky modifikované rostliny MeSH
- homozygot MeSH
- klíčení MeSH
- květy fyziologie MeSH
- molekulární chaperony genetika metabolismus MeSH
- mutace MeSH
- proteiny huseníčku genetika metabolismus MeSH
- proteomika metody MeSH
- regulace genové exprese u rostlin MeSH
- semena rostlinná MeSH
- transkriptom MeSH
- vývoj rostlin * genetika MeSH
- Publikační typ
- časopisecké články MeSH
Reproduction success in angiosperm plants depends on robust pollen tube growth through the female pistil tissues to ensure successful fertilization. Accordingly, there is an apparent evolutionary trend to accumulate significant reserves during pollen maturation, including a population of stored mRNAs, that are utilized later for a massive translation of various proteins in growing pollen tubes. Here, we performed a thorough transcriptomic and proteomic analysis of stored and translated transcripts in three subcellular compartments of tobacco (Nicotiana tabacum), long-term storage EDTA/puromycin-resistant particles, translating polysomes, and free ribonuclear particles, throughout tobacco pollen development and in in vitro-growing pollen tubes. We demonstrated that the composition of the aforementioned complexes is not rigid and that numerous transcripts were redistributed among these complexes during pollen development, which may represent an important mechanism of translational regulation. Therefore, we defined the pollen sequestrome as a distinct and highly dynamic compartment for the storage of stable, translationally repressed transcripts and demonstrated its dynamics. We propose that EDTA/puromycin-resistant particle complexes represent aggregated nontranslating monosomes as the primary mediators of messenger RNA sequestration. Such organization is extremely useful in fast tip-growing pollen tubes, where rapid and orchestrated protein synthesis must take place in specific regions.
- MeSH
- polyribozomy genetika metabolismus MeSH
- proteom genetika metabolismus MeSH
- proteomika metody MeSH
- pyl genetika růst a vývoj metabolismus MeSH
- pylová láčka genetika růst a vývoj metabolismus MeSH
- regulace genové exprese u rostlin MeSH
- ribonukleoproteiny genetika metabolismus MeSH
- ribozomy genetika metabolismus MeSH
- rostlinné proteiny genetika metabolismus MeSH
- stanovení celkové genové exprese metody MeSH
- tabák genetika růst a vývoj metabolismus MeSH
- vývojová regulace genové exprese MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Protein phosphorylation was repeatedly shown to be the most dynamic post-translational modification mediated by a huge orchestra of protein kinases and phosphatases. Upon landing on a stigma, pollen grain dehydration and activation are accompanied by changes in protein phosphorylation together with the translation activation of stored mRNAs. To enable studies of the total phosphoproteome, it is usually necessary to apply various enrichment techniques. In this chapter, one of these protocols that worked previously well on tobacco mature pollen is presented in more detail. The method comprises of three basic steps: (1) picking flowers from the flowering tobacco plants (Nicotiana tabacum cv. Samsun), and collection of the shed pollen grains; (2) extraction of total proteins by TCA/acetone; (3) phosphoprotein enrichment by MOAC with aluminum hydroxide matrix. Taken together this protocol describes how to isolate phosphoproteins out of tobacco mature pollen.
- MeSH
- chromatografie afinitní MeSH
- fosfoproteiny chemie izolace a purifikace metabolismus MeSH
- hydroxid hlinitý chemie MeSH
- posttranslační úpravy proteinů MeSH
- pyl metabolismus MeSH
- rostlinné proteiny chemie izolace a purifikace metabolismus MeSH
- tabák metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
Průhledy ; sv. XVI
Vydání první 187 stran : ilustrace (převážně barevné), tabulky ; 21 cm
- MeSH
- biologická evoluce MeSH
- ekologie MeSH
- rostliny MeSH
- rozmnožování MeSH
- sexualita MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- populární práce MeSH
- Konspekt
- Obecná biologie
- NLK Obory
- biologie
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
KEY MESSAGE: Overview of pollen development. Male gametophyte development of angiosperms is a complex process that requires coordinated activity of different cell types and tissues of both gametophytic and sporophytic origin and the appropriate specific gene expression. Pollen ontogeny is also an excellent model for the dissection of cellular networks that control cell growth, polarity, cellular differentiation and cell signaling. This article describes two sequential phases of angiosperm pollen ontogenesis-developmental phase leading to the formation of mature pollen grains, and a functional or progamic phase, beginning with the impact of the grains on the stigma surface and ending at double fertilization. Here we present an overview of important cellular processes in pollen development and explosive pollen tube growth stressing the importance of reserves accumulation and mobilization and also the mutual activation of pollen tube and pistil tissues, pollen tube guidance and the communication between male and female gametophytes. We further describe the recent advances in regulatory mechanisms involved such as posttranscriptional regulation (including mass transcript storage) and posttranslational modifications to modulate protein function, intracellular metabolic signaling, ionic gradients such as Ca(2+) and H(+) ions, cell wall synthesis, protein secretion and intercellular signaling within the reproductive tissues.
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.
