Pollen Translation Group
The progamic phase of male gametophyte development involves activation of synthetic and catabolic processes required for the rapid growth of the pollen tube. It is well-established that both transcription and translation play an important role in global and specific gene expression patterns during pollen maturation. On the contrary, germination of many pollen species has been shown to be largely independent of transcription but vitally dependent on translation of stored mRNAs. We published the first structural and proteomic data about large ribonucleoprotein particles (EPP granules) in tobacco male gametophyte. These complexes are formed in immature pollen where they contain translationally silent mRNAs. Although massively activated at the early progamic phase, they also serve as a long-term storage of mRNA transported along with the translational machinery to the tip region. Moreover, EPPs were shown to contain ribosomal subunits, rRNAs and a set of mRNAs. Presented results extend our view of EPP complexes from mere RNA storage and transport compartment in particular stages of pollen development to the complex and well-organized machinery devoted to mRNA storage, transport and subsequent controlled activation resulting in protein synthesis, processing and precise localization. Such an organization is extremely useful in fast tip-growing pollen tube. There, massive and orchestrated protein synthesis, processing, and transport must take place in accurately localized regions. Moreover, presented complex role of EPPs in tobacco cytoplasmic mRNA and protein metabolism makes them likely to be active in another plant species too. Expression of vast majority of the closest orthologs of EPP proteins also in Arabidopsis male gametophyte further extends this concept from tobacco to Arabidopsis, the model species with advanced tricellular pollen.
Recently, we have initiated new direction of our research, tobacco pollen translatomics. It has been well established that both transcription and translation play an important role in global and specific gene expression patterns during pollen maturation. On the contrary, germination of many pollen species has been shown to be largely independent of transcription but vitally dependent on translation of stored mRNAs.
We demonstrated that large ribuncleoprotein particles (EPP granules) contained translationally silent mRNAs transported along with the translational machinery to the tip region where the translation took place. Such an organization is extremely useful in fast tip-growing pollen tube. Moreover, the asymmetric mRNA distribution is the determinant of protein gradient influencing cell polarity, cell fate and overall patterning during development. we proposed a model outlining the network of post-transcriptional control with a focus on the role of stored RNPs and started the functional characterization of RNA-binding proteins. We have extended our transcriptomic and proteomic analyses to cover three cytoplasmic subfractions containing mRNAs at different translational status and to demonstrate their developmental dynamics – 1) actively translated transcripts associated with polysomes (PS - termed translatome), 2) pollen mRNAs bound to pollen stored ribonucleoprotein particles (stored mRNPs/free mRNPs - termed mRNPome) and 3) long-term stored transcripts on EPP granules (EPPs - termed sequestrome).
Tobacco pollen developmental phosphoproteomics
Rapid changes of protein phosphorylation play a crucial role in the regulation of many cellular processes and therefore protein phosphorylation dynamics during pollen grain activation on stigma attracted our attention. Being post-translationally modified, phosphoproteins are often present in low abundance and tend to co-exist with their unphosphorylated isoforms within the cell. Therefore we first developed the protein extraction protocol suitable for subsequent phosphoprotein enrichment from tough tobacco pollen and selected the appropriate phosphopeptide enrichment procedure (MOAC) including the general review of phosphoprotein and phosphopeptide enrichment protocols. We hypothesized that the transition between quiescent mature and the metabolically active germinating pollen grain involved changes in protein phosphorylation. Therefore we have used metal oxide/hydroxide affinity chromatography (MOAC) based on an aluminium hydroxide matrix to generate a population of phosphoprotein candidates from both mature and in vitro activated tobacco pollen grains. Both in-gel and gel-free methods allied to MS were applied to identify a set of 139 phosphoprotein candidates, some of them were validated by in vitro phosphorylation including the detection of 52 phosphorylation sites. As a joint effort with Dr. H.-P. Mock’s (IPK Gatersleben) group, we showed for the first time the dynamics of protein phosphorylation and dephosphorylation associated with early stages of pollen germination.
The tobacco phosphoproteome dataset was compared to previously published Arabidopsis mature pollen phosphoproteome (Mayank et al. 2012, Plant J 72: 89-101). The representation of the O-phosphorylated amino acids was evaluated between two species and the putative pollen-specific or pollen-abundant phosphopeptides were highlighted. Finally, the phosphorylation sites common for both Arabidopsis and tobacco phosphoproteins were listed as well as the phosphorylation motifs identified.
In collaboration with H.-P. Mock’s (IPK Gatersleben, Germany) group, we finely tuned this analysis to three time points – mature pollen, 5-min and 30-min-activated pollen. We identified 471 phosphopeptides (301 phosphoproteins) carrying 432 phosphorylation sites, position of which was exactly matched by mass spectrometry. The majority of differentially phosphorylated proteins fell into GO categories clearly connected to pollen activation with the re-initiation of transcription and translation. The quantitative data highlighted the regulatory trends; we showed that several phosphopeptides representing the same phosphoprotein underwent different regulation, which pinpointed the complexity and dynamics of protein phosphorylation at the initiation of the progamic phase. Collectively, we showed the first phosphoproteomics data on activated pollen where the position of the respective phosphorylation sites was clearly demonstrated.
Tobacco pollen secretomics
Some of the stored mRNAs may encode for secreted proteins required for male-female signalling during pollen tube guidance. To understand the spectrum of translational regulation and mRNA storage, we studied pollen tube secretomics as “bottom-up” approach to link with our sequestrome transcriptome. It is established that 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) in collaboration with M. Johnson, Brown University, USA and R. Palanivelu, Univ. of Arizona, USA. As a joint effort with Z. Zdráhal’s group (CEITEC MU, Brno), we performed gel-free LC-MS/MS for high throughput analysis of pollen-tube-secreted proteins. Our approach has led to the identification of over 341 protein groups on average (801 accessions). Among them are pollen tube-secreted ligands and receptor proteins representing potential male components in perceiving ovule-emitted cues for guidance. This study has uncovered novel pistil-dependent pollen tube-secreted proteins critical for establishing male-female signalling interaction map for successful sperm cells delivery and fertilization and as means to overcome interspecies pre-zygotic barriers.