Sexual reproduction in angiosperms requires the production and delivery of two male gametes by a three-celled haploid male gametophyte. This demands synchronized gene expression in a short developmental window to ensure double fertilization and seed set. While transcriptomic changes in developing pollen are known for Arabidopsis, no studies have integrated RNA and proteomic data in this model. Further, the role of alternative splicing has not been fully addressed, yet post-transcriptional and post-translational regulation may have a key role in gene expression dynamics during microgametogenesis. We have refined and substantially updated global transcriptomic and proteomic changes in developing pollen for two Arabidopsis accessions. Despite the superiority of RNA-seq over microarray-based platforms, we demonstrate high reproducibility and comparability. We identify thousands of long non-coding RNAs as potential regulators of pollen development, hundreds of changes in alternative splicing and provide insight into mRNA translation rate and storage in developing pollen. Our analysis delivers an integrated perspective of gene expression dynamics in developing Arabidopsis pollen and a foundation for studying the role of alternative splicing in this model.

• Klíčová slova
• Arabidopsis, Male gametophyte, Microgametogenesis, Proteome, RNA-seq,
• MeSH
• Arabidopsis * genetika   metabolismus   MeSH
• proteiny huseníčku * genetika   metabolismus   MeSH
• proteomika MeSH
• pyl genetika   metabolismus   MeSH
• regulace genové exprese u rostlin MeSH
• reprodukovatelnost výsledků MeSH
• transkriptom MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteiny huseníčku * MeSH

Being rooted in place, plants are faced with the challenge of responding to unfavourable local conditions. One such condition, heat stress, contributes massively to crop losses globally. Heatwaves are predicted to increase, and it is of vital importance to generate crops that are tolerant to not only heat stress but also to several other abiotic stresses (e.g. drought stress, salinity stress) to ensure that global food security is protected. A better understanding of the molecular mechanisms that underlie the temperature stress response in pollen will be a significant step towards developing effective breeding strategies for high and stable production in crop plants. While most studies have focused on the vegetative phase of plant growth to understand heat stress tolerance, it is the reproductive phase that requires more attention as it is more sensitive to elevated temperatures. Every phase of reproductive development is affected by environmental challenges, including pollen and ovule development, pollen tube growth, male-female cross-talk, fertilization, and embryo development. In this review we summarize how pollen is affected by heat stress and the molecular mechanisms employed during the stress period, as revealed by classical and -omics experiments.

• Klíčová slova
• heat stress (HS), heat stress response (HSR), multiomics, pollen development, thermotolerance,
• MeSH
• fyziologický stres MeSH
• pyl MeSH
• reakce na tepelný šok MeSH
• šlechtění rostlin * MeSH
• termotolerance * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

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.

• Klíčová slova
• Flowering plants, Male gametophyte, Pollen development, Pollen tube growth,
• MeSH
• Magnoliopsida růst a vývoj   metabolismus   MeSH
• pyl růst a vývoj   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy 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
• Názvy látek
• fosfoproteiny MeSH
• rostlinné proteiny MeSH

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.

• MeSH
• barvení a značení MeSH
• chromatografie afinitní MeSH
• fosfoproteiny chemie   izolace a purifikace   metabolismus   MeSH
• fosforylace MeSH
• hmotnostní spektrometrie MeSH
• imunoprecipitace MeSH
• peptidové fragmenty chemie   izolace a purifikace   metabolismus   MeSH
• posttranslační úpravy proteinů MeSH
• proteom chemie   izolace a purifikace   metabolismus   MeSH
• proteomika MeSH
• rostlinné proteiny chemie   izolace a purifikace   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• fosfoproteiny MeSH
• peptidové fragmenty MeSH
• proteom MeSH
• rostlinné proteiny MeSH