Gene expression regulation during tissue development is extremely complex. A key mechanism of gene regulation is the recognition of regulatory motifs, also known as cis-regulatory elements (CREs), by various proteins in gene promoter regions. Localization of these motifs near the transcription start site (TSS) or translation start site (ATG) is crucial for transcription initiation and rate. Transcription levels of individual genes, regulated by these motifs, can vary significantly across tissues and developmental stages, especially in processes like sexual reproduction. However, the precise localization and visualization of these motifs in relation to gene expression in specific tissues can be challenging. Here, we introduce a freely available tool called GOLEM (Gene regulatOry eLEMents; https://golem.ncbr.muni.cz), which enables users to precisely locate any motif of interest with respect to TSS or ATG within the relevant plant genomes across the plant Tree of Life (Chara, Marchantia, Physcomitrium, Azolla, Ceratopteris, Amborella, Oryza, Zea, Solanum and Arabidopsis). The visualization of the motifs is performed with respect to the transcript levels of particular genes in leaves and male reproductive tissues and can be compared with genome-wide distribution regardless of the transcription level. Additionally, genes with specific CREs at defined positions and high expression in selected tissues can be exported for further analysis. GOLEM's functionality is illustrated by its application to conserved motifs (e.g. TATA-box, ABRE, I-box, and TC-element), hormone-responsive elements (GCC-box, ARR10_binding motif), as well as to male gametophyte-related motifs (e.g., LAT52, MEF2, and DOF_core).

• Klíčová slova
• GOLEM, Gene regulatOry eLEMents, TSS, gametophyte, motif localization, plant genes, promoter elements, technical advance,
• MeSH
• Arabidopsis genetika   MeSH
• genom rostlinný genetika   MeSH
• počátek transkripce MeSH
• promotorové oblasti (genetika) * genetika   MeSH
• pyl * genetika   MeSH
• regulace genové exprese u rostlin genetika   MeSH
• software * MeSH
• Publikační typ
• časopisecké články MeSH

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

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.

• Klíčová slova
• kinase motif, male gametophyte, phosphoproteomics, pollen tube, root hair, signal transduction,
• MeSH
• fosfoproteiny metabolismus   MeSH
• opylení * MeSH
• proteom metabolismus   MeSH
• proteomika * MeSH
• pylová láčka metabolismus   MeSH
• rostlinné proteiny metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• fosfoproteiny MeSH
• proteom MeSH
• rostlinné proteiny MeSH

Viroids are small, non-coding, parasitic RNAs that promote developmental distortions in sensitive plants. We analyzed pollen of Nicotiana benthamiana after infection and/or ectopic transformation with cDNAs of citrus bark cracking viroid (CBCVd), apple fruit crinkle viroid (AFCVd) and potato spindle tuber viroid (PSTVd) variant AS1. These viroids were seed non-transmissible in N. benthamiana. All viroids propagated to high levels in immature anthers similar to leaves, while their levels were drastically reduced by approximately 3.6 × 103, 800 and 59 times in mature pollen of CBCVd, AFCVd and PSTVd infected N. benthamiana, respectively, in comparison to leaves. These results suggest similar elimination processes during male gametophyte development as in the Nicotiana tabacum we presented in our previous study. Mature pollen of N. benthamiana showed no apparent defects in infected plants although all three viroids induced strong pathological symptoms on leaves. While Nicotiana species have naturally bicellular mature pollen, we noted a rare occurrence of mature pollen with three nuclei in CBCVd-infected N. benthamiana. Changes in the expression of ribosomal marker proteins in AFCVd-infected pollen were detected, suggesting some changes in pollen metabolism. N. benthamiana transformed with 35S-driven viroid cDNAs showed strong symptoms including defects in pollen development. A large number of aborted pollen (34% and 62%) and a slight increase of young pollen grains (8% and 15%) were found in mature pollen of AFCVd and CBCVd transformants, respectively, in comparison to control plants (3.9% aborted pollen and 0.3% young pollen). Moreover, pollen grains with malformed nuclei or trinuclear pollen were found in CBCVd-transformed plants. Our results suggest that "forcing" overexpression of seed non-transmissible viroid led to strong pollen pathogenesis. Viroid adaptation to pollen metabolism can be assumed as an important factor for viroid transmissibility through pollen and seeds.

• Klíčová slova
• AFCVd and PSTVd parasitic RNAs, CBCVd, Humulus lupulus, Nicotiana benthamiana, male gametophyte, plant transformation, proteomic of viroid infected pollen, viroid elimination,
• Publikační typ
• časopisecké články MeSH

Increasing evidence suggests that posttranscriptional regulation is a key player in the transition between mature pollen and the progamic phase (from pollination to fertilization). Nonetheless, the actors in this messenger RNA (mRNA)-based gene expression reprogramming are poorly understood. We demonstrate that the evolutionarily conserved RNA-binding protein LARP6C is necessary for the transition from dry pollen to pollen tubes and the guided growth of pollen tubes towards the ovule in Arabidopsis thaliana. In dry pollen, LARP6C binds to transcripts encoding proteins that function in lipid synthesis and homeostasis, vesicular trafficking, and polarized cell growth. LARP6C also forms cytoplasmic granules that contain the poly(A) binding protein and possibly represent storage sites for translationally silent mRNAs. In pollen tubes, the loss of LARP6C negatively affects the quantities and distribution of storage lipids, as well as vesicular trafficking. In Nicotiana benthamiana leaf cells and in planta, analysis of reporter mRNAs designed from the LARP6C target MGD2 provided evidence that LARP6C can shift from a repressor to an activator of translation when the pollen grain enters the progamic phase. We propose that LARP6C orchestrates the timely posttranscriptional regulation of a subset of mRNAs in pollen during the transition from the quiescent to active state and along the progamic phase to promote male fertilization in plants.

• MeSH
• 5' nepřekládaná oblast MeSH
• Arabidopsis cytologie   genetika   růst a vývoj   MeSH
• cytoplazmatická granula genetika   metabolismus   MeSH
• geneticky modifikované rostliny MeSH
• lipidy biosyntéza   genetika   MeSH
• messenger RNA genetika   metabolismus   MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• proteiny vázající RNA genetika   metabolismus   MeSH
• pylová láčka cytologie   genetika   růst a vývoj   MeSH
• regulace genové exprese u rostlin MeSH
• RNA rostlin metabolismus   MeSH
• tabák genetika   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 5' nepřekládaná oblast MeSH
• AT3G19090 protein, Arabidopsis MeSH Prohlížeč
• lipidy MeSH
• messenger RNA MeSH
• proteiny huseníčku MeSH
• proteiny vázající RNA MeSH
• RNA rostlin 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

ALBA DNA/RNA-binding proteins form an ancient family, which in eukaryotes diversified into two Rpp25-like and Rpp20-like subfamilies. In most studied model organisms, their function remains unclear, but they are usually associated with RNA metabolism, mRNA translatability and stress response. In plants, the enriched number of ALBA family members remains poorly understood. Here, we studied ALBA dynamics during reproductive development in Arabidopsis at the levels of gene expression and protein localization, both under standard conditions and following heat stress. In generative tissues, ALBA proteins showed the strongest signal in mature pollen where they localized predominantly in cytoplasmic foci, particularly in regions surrounding the vegetative nucleus and sperm cells. Finally, we demonstrated the involvement of two Rpp25-like subfamily members ALBA4 and ALBA6 in RNA metabolism in mature pollen supported by their co-localization with poly(A)-binding protein 3 (PABP3). Collectively, we demonstrated the engagement of ALBA proteins in male reproductive development and the heat stress response, highlighting the involvement of ALBA4 and ALBA6 in RNA metabolism, storage and/or translational control in pollen upon heat stress. Such dynamic re-localization of ALBA proteins in a controlled, developmentally and environmentally regulated manner, likely reflects not only their redundancy but also their possible functional diversification in plants.

• Klíčová slova
• ALBA, Arabidopsis thaliana, PABP3, confocal microscopy, expression analysis, flowering, heat stress, pollen development, protein localization,
• MeSH
• Arabidopsis embryologie   metabolismus   MeSH
• fyziologický stres genetika   MeSH
• konfokální mikroskopie MeSH
• květy růst a vývoj   MeSH
• poly(A)-vazebné proteiny metabolismus   MeSH
• promotorové oblasti (genetika) genetika   MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• proteiny vázající RNA genetika   metabolismus   MeSH
• pyl embryologie   MeSH
• reakce na tepelný šok fyziologie   MeSH
• regulace genové exprese u rostlin genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• poly(A)-vazebné proteiny MeSH
• proteiny huseníčku MeSH
• proteiny vázající RNA MeSH

Flowering plants (angiosperms) are characterized by pollen tubes (PTs; male gametophytes) carrying two immobile sperm cells that grow over long distances through the carpel toward the ovules, where double fertilization is executed. It is not understood how these reproductive structures evolved, which genes occur de novo in male gametophytes of angiosperms, and to which extent PT functions are conserved among angiosperms. To contribute to a deeper understanding of the evolution of gametophyte functions, we generated RNA sequencing data from seven reproductive and two vegetative control tissues of the basal angiosperm Amborella trichopoda and complemented these with proteomic data of pollen grains (PGs) and PTs. The eudicot model plant Arabidopsis (Arabidopsis thaliana) served as a reference organism for data analysis, as more than 200 genes have been associated with male gametophyte functions in this species. We describe methods to collect bicellular A. trichopoda PGs, to induce their germination in vitro, and to monitor PT growth and germ cell division. Transcriptomic and proteomic analyses indicate that A. trichopoda PGs are prepared for germination requiring lipids, energy, but likely also reactive oxygen species, while PTs are especially characterized by catabolic/biosynthetic and transport processes including cell wall biosynthesis and gene regulation. Notably, a number of pollen-specific genes were lacking in Arabidopsis, and the number of genes involved in pollen signaling is significantly reduced in A. trichopoda In conclusion, we provide insight into male gametophyte functions of the most basal angiosperm and establish a valuable resource for future studies on the evolution of flowering plants.

• MeSH
• Arabidopsis genetika   růst a vývoj   MeSH
• biologická evoluce MeSH
• klíčení genetika   fyziologie   MeSH
• Magnoliopsida genetika   růst a vývoj   MeSH
• proteomika MeSH
• pyl genetika   růst a vývoj   MeSH
• pylová láčka genetika   růst a vývoj   MeSH
• regulace genové exprese u rostlin MeSH
• rostlinné geny MeSH
• transkriptom MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Hydrogen can serve as an electron donor for chemolithotrophic acidophiles, especially in the deep terrestrial subsurface and geothermal ecosystems. Nevertheless, the current knowledge of hydrogen utilization by mesophilic acidophiles is minimal. A multi-omics analysis was applied on Acidithiobacillus ferrooxidans growing on hydrogen, and a respiratory model was proposed. In the model, [NiFe] hydrogenases oxidize hydrogen to two protons and two electrons. The electrons are used to reduce membrane-soluble ubiquinone to ubiquinol. Genetically associated iron-sulfur proteins mediate electron relay from the hydrogenases to the ubiquinone pool. Under aerobic conditions, reduced ubiquinol transfers electrons to either cytochrome aa 3 oxidase via cytochrome bc 1 complex and cytochrome c 4 or the alternate directly to cytochrome bd oxidase, resulting in proton efflux and reduction of oxygen. Under anaerobic conditions, reduced ubiquinol transfers electrons to outer membrane cytochrome c (ferrireductase) via cytochrome bc 1 complex and a cascade of electron transporters (cytochrome c 4, cytochrome c 552, rusticyanin, and high potential iron-sulfur protein), resulting in proton efflux and reduction of ferric iron. The proton gradient generated by hydrogen oxidation maintains the membrane potential and allows the generation of ATP and NADH. These results further clarify the role of extremophiles in biogeochemical processes and their impact on the composition of the deep terrestrial subsurface.

• Klíčová slova
• Acidithiobacillus, extremophiles, ferric iron reduction, hydrogen metabolism, multi-omics, oxygen reduction,
• Publikační typ
• časopisecké články MeSH

Tobacco (Nicotiana tabacum) pollen is a well-suited model for studying many fundamental biological processes owing to its well-defined and distinct development stages. It is also one of the major agents involved in the transmission of infectious viroids, which is the primary mechanism of viroid pathogenicity in plants. However, some viroids are non-transmissible and may be possibly degraded or eliminated during the gradual process of pollen development maturation. The molecular details behind the response of developing pollen against the apple fruit crinkle viroid (AFCVd) infection and viroid eradication is largely unknown. In this study, we performed an integrative analysis of the transcriptome and proteome profiles to disentangle the molecular cascade of events governing the three pollen development stages: early bicellular pollen (stage 3, S3), late bicellular pollen (stage 5, S5), and 6 h-pollen tube (PT6). The integrated analysis delivered the molecular portraits of the developing pollen against AFCVd infection, including mechanistic insights into the viroid eradication during the last steps of pollen development. The isobaric tags for label-free relative quantification (iTRAQ) with digital gene expression (DGE) experiments led us to reliably identify subsets of 5321, 5286, and 6923 proteins and 64,033, 60,597, and 46,640 expressed genes in S3, S5, and PT6, respectively. In these subsets, 2234, 2108 proteins and 9207 and 14,065 mRNAs were differentially expressed in pairwise comparisons of three stages S5 vs. S3 and PT6 vs. S5 of control pollen in tobacco. Correlation analysis between the abundance of differentially expressed mRNAs (DEGs) and differentially expressed proteins (DEPs) in pairwise comparisons of three stages of pollen revealed numerous discordant changes in mRNA/protein pairs. Only a modest correlation was observed, indicative of divergent transcription, and its regulation and importance of post-transcriptional events in the determination of the fate of early and late pollen development in tobacco. The functional and enrichment analysis of correlated DEGs/DEPs revealed the activation in pathways involved in carbohydrate metabolism, amino acid metabolism, lipid metabolism, and cofactor as well as vitamin metabolism, which points to the importance of these metabolic pathways in pollen development. Furthermore, the detailed picture of AFCVd-infected correlated DEGs/DEPs was obtained in pairwise comparisons of three stages of infected pollen. The AFCVd infection caused the modulation of several genes involved in protein degradation, nuclear transport, phytohormone signaling, defense response, and phosphorylation. Intriguingly, we also identified several factors including, DNA-dependent RNA-polymerase, ribosomal protein, Argonaute (AGO) proteins, nucleotide binding proteins, and RNA exonucleases, which may plausibly involve in viroid stabilization and eradication during the last steps of pollen development. The present study provides essential insights into the transcriptional and translational dynamics of tobacco pollen, which further strengthens our understanding of plant-viroid interactions and support for future mechanistic studies directed at delineating the functional role of candidate factors involved in viroid elimination.

• Klíčová slova
• AFCVd propagation and eradication, Nicotiana tabacum, Proteome, RNA sequencing, RT qPCR, male gametophyte, viroid degradation, viroid replication,
• MeSH
• buněčná diferenciace * MeSH
• nemoci rostlin virologie   MeSH
• proteomika * MeSH
• pyl * metabolismus   virologie   MeSH
• rostlinné viry metabolismus   MeSH
• stanovení celkové genové exprese * MeSH
• tabák * metabolismus   virologie   MeSH
• viroidy metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH