Jan Fíla

Enrichment techniques employed in phosphoproteomics

Rapid changes of protein phosphorylation play a crucial role in the regulation of many cellular processes. Being post-translationally modified, phosphoproteins are often present in quite low abundance and tend to co-exist with their unphosphorylated isoform within the cell. To make their identification more practicable, the use of enrichment protocols is often required. The enrichment strategies can be performed either at the level of phosphoproteins or at the level of phosphopeptides. Both approaches have their advantages and disadvantages. Most enriching strategies are based on chemical modifications, affinity chromatography to capture peptides and proteins containing negatively charged phosphate groups onto a positively charged matrix, or immunoprecipitation by phospho-specific antibodies.In this article, the most up-to-date enrichment techniques are discussed, taking into account their optimization, and highlighting their advantages and disadvantages. Moreover, these methods are compared to each other, revealing their complementary nature in providing comprehensive coverage of the phosphoproteome.

Male gametophyte development and function in angiosperms: a general concept

Key messageOverview of pollen development.AbstractMale 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 Ca2+ and H+ ions, cell wall synthesis, protein secretion and intercellular signaling within the reproductive tissues.

Revealing phosphoproteins playing role in tobacco pollen activated in vitro

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.

Phosphoproteomics profiling of tobacco mature pollen and pollen activated in vitro

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.

In search of ligands and receptors of the pollen tube: the missing link in pollen tube perception

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.

Impact of homogenization and protein extraction conditions on the obtained tobacco pollen proteomic patterns

Mature pollen grain represents a highly desiccated structure with an extremely tough cell wall. Thanks to it, it resists common proteomic protocols. Instead, a robust homogenization has to be performed since proteins are needed to burst out of the cell to be included in the extracted proteome fraction. Here, a novel way of pollen homogenization employing Roche MagNA Lyser Instrument is presented, sparing time and laborious work. However, plant proteomics does not rely solely on perfect homogenization; also the choice of the extraction protocol is of key importance. The composition of the extraction buffer has a decisive influence on which proteome fraction will be extracted. Therefore the second part of our study is dedicated to the comparison of different extraction protocols with respect to subsequent proteomic analyses.

Phosphoproteomic studies in Arabidopsis and tobacco male gametophytes

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.

Dynamics of the Pollen Sequestrome Defined by Subcellular Coupled Omics

The pollen sequestrome is found to be a highly dynamic compartment for the storage of translationally repressed mRNAs, and nontranslating monosomes are proposed to be the primary mediators of mRNA sequestration in Nicotiana tabacum. 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.

Phosphoprotein Enrichment from Tobacco Mature Pollen Crude Protein Extract

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.

When Simple Meets Complex: Pollen and the -Omics

Pollen, an extremely reduced bi-cellular or tri-cellular male reproductive structure of flowering plants, serves as a model for numerous studies covering a wide range of developmental and physiological processes. The pollen development and subsequent progamic phase represent two fragile and vital phases of plant ontogenesis, and pollen was among the first singular plant tissues thoroughly characterised at the transcriptomic level. Here we present an overview of high-throughput tools applied in pollen research on numerous plant species. Transcriptomics, being the first experimental approach used, has provided and continues providing valuable information about global and specific gene expression and its dynamics. However, the proteome does not fully reflect the transcriptome, namely, because post-transcriptional regulatory levels, especially translation, mRNA storage and protein modifications, are active during male gametophyte development and during progamic phase. Transcriptomics therefore should be complemented by other -omic tools to get more realistic insight, most importantly proteomics and other specialised approaches mapping the involvement of regulatory RNAs and protein post-translational modifications as well as experiments designed to identify the subsets of total -omes like translatome, secretome or allergome.