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.
Heat shock proteins 90 (HSP90) are essential and play critical roles in the adaptation of organisms to diverse stimuli. In plants, HSP90 are involved in auxin, jasmonate and brassinosteroid (BR) signalling pathways. The BR-promoted activation of the BES1 transcription factor regulates BR-responsive genes. Using genetic, physiological, fluorescence live cell imaging, molecular and biochemical approaches, such as phenotypic analysis, co-immunoprecipitation assay, yeast-two hybrid and Bimolecular fluorescence complementation (BiFC), we studied complex formation between BES1 and HSP90 under control conditions and active BR signalling. Further, we determined the effect of the pharmacological inhibition of HSP90 ATPase activity on hypocotyl elongation of bes1-D mutant. We determined that HSP90 interact with BES1 in the nucleus and in the cytoplasm. During active BR signalling, nuclear complexes were absent while cytoplasmic HSP90/BES1 complexes were prominent. Our results showed that the hypocotyl length of bes1-D mutants was highly reduced when HSP90 was challenged by the geldanamycin (GDA) inhibitor of the ATPase activity of HSP90. Active BR signalling could not rescue the GDA effect on the hypocotyl elongation of bes1-D. Our results reveal that the constitutively active BES1 in the bes1-D mutant is hypersensitive to GDA. The interaction of HSP90 with BES1 argues that HSP90 facilitate the nuclear metastable conformation of BES1 to regulate BR-dependent gene expression, and our data show that HSP90 assist in the compartmentalised cycle of BES1 during active BR signalling.
- MeSH
- Arabidopsis * MeSH
- brassinosteroidy * metabolismus MeSH
- DNA vazebné proteiny metabolismus MeSH
- proteiny huseníčku * metabolismus MeSH
- proteiny tepelného šoku HSP90 * metabolismus MeSH
- regulace genové exprese u rostlin MeSH
- signální transdukce * MeSH
- tabák metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
Formins are evolutionarily conserved multi-domain proteins participating in the control of both actin and microtubule dynamics. Angiosperm formins form two evolutionarily distinct families, Class I and Class II, with class-specific domain layouts. The model plant Arabidopsis thaliana has 21 formin-encoding loci, including 10 Class II members. In this study, we analyze the subcellular localization of two A. thaliana Class II formins exhibiting typical domain organization, the so far uncharacterized formin AtFH13 (At5g58160) and its distant homolog AtFH14 (At1g31810), previously reported to bind microtubules. Fluorescent protein-tagged full length formins and their individual domains were transiently expressed in Nicotiana benthamiana leaves under the control of a constitutive promoter and their subcellular localization (including co-localization with cytoskeletal structures and the endoplasmic reticulum) was examined using confocal microscopy. While the two formins exhibit distinct and only partially overlapping localization patterns, they both associate with microtubules via the conserved formin homology 2 (FH2) domain and with the periphery of the endoplasmic reticulum, at least in part via the N-terminal PTEN (Phosphatase and Tensin)-like domain. Surprisingly, FH2 domains of AtFH13 and AtFH14 can form heterodimers in the yeast two-hybrid assay-a first case of potentially biologically relevant formin heterodimerization mediated solely by the FH2 domain.
- MeSH
- Arabidopsis genetika metabolismus MeSH
- dimerizace MeSH
- endoplazmatické retikulum metabolismus MeSH
- exprese genu MeSH
- forminy genetika metabolismus MeSH
- mikrotubuly metabolismus MeSH
- proteinové domény MeSH
- proteiny huseníčku genetika metabolismus MeSH
- rekombinantní proteiny genetika metabolismus MeSH
- tabák metabolismus MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
Selenium (Se) is a natural trace element, which shifts its action in a relatively narrow concentration range from nutritional role to toxicity. Although it has been well established that in plants chloroplasts are among the primary targets, the mechanism of toxicity on photosynthesis is not well understood. Here, we compared selenate and red-allotrope elemental selenium nanoparticles (red nanoSe) in in vitro tobacco cultures to investigate their effects on the structure and functions of the photosynthetic machinery. Selenate at 10 mg/L concentration retarded plant growth; it also led to a decreased chlorophyll content, accompanied with an increase in the carotenoid-to-chlorophyll ratio. Structural examinations of the photosynthetic machinery, using electron microscopy, small-angle neutron scattering and circular dichroism spectroscopy, revealed significant perturbation in the macro-organization of the pigment-protein complexes and sizeable shrinkage in the repeat distance of granum thylakoid membranes. As shown by chlorophyll a fluorescence transient measurements, these changes in the ultrastructure were associated with a significantly diminished photosystem II activity and a reduced performance of the photosynthetic electron transport, and an enhanced capability of non-photochemical quenching. These changes in the structure and function of the photosynthetic apparatus explain, at least in part, the retarded growth of plantlets in the presence of 10 mg/L selenate. In contrast, red nanoSe, even at 100 mg/L and selenate at 1 mg/L, exerted no negative effect on the growth of plantlets and affected only marginally the thylakoid membrane ultrastructure and the photosynthetic functions.
Growth and developmental changes in plants induced by pharmaceuticals reflect changes in processes at the cellular and subcellular levels. Due to their growth and cellular characteristics, plant cell suspension cultures can be a suitable model for assessing toxicity. In this study, 10-1000 μg/L of the non-steroidal anti-inflammatory drug diclofenac (DCF) decreased the viability of Nicotiana tabacum BY-2 cells after 24 h of treatment. Further, 0.1-10 mg/L DCF diminished the density of the cell suspension by 9-46% after 96 h of treatment, but at 1 and 10 μg/L, DCF increased the density by 13% and 5%, respectively, after 120 h. These changes were accompanied by increased production of total reactive oxygen species (ROS) and mitochondrial superoxide (up to 17-fold and 5-fold, respectively), and a decrease in the mitochondrial membrane potential (by ∼64%) especially at 1000 μg/L DCF. The increased ROS production was accompanied by decrease in level of reactive nitrogen species (RNS; by 36%) and total thiols (by 61%). Damage to BY-2 cells was evidenced by accumulation of neutral red in acidic compartments (up to 10-fold at 1000 μg/L DCF), and increase of autophagic vacuole formation (up to 8-fold at 1000 μg/L DCF). Furthermore, irregular or stretched nuclei were observed in nearly 27% and 50% of cells at 100 and 1000 μg/L DCF, respectively. Highest levels of chromatin condensation (11% of cells) and apoptotic DNA fragmentation (7%) were found at 10 μg/L DCF. The results revealed a significant effect of DCF on BY-2 cells after 24 h of exposure. Changes in the growth and viability parameters were indisputably related to ROS and RNS production, changes in mitochondrial function, and possible activation of processes leading to cell death.
WEE1 regulates the cell cycle by inactivating cyclin dependent protein kinases (CDKs) via phosphorylation. In yeast and animal cells, CDC25 phosphatase dephosphorylates the CDK releasing cells into mitosis, but in plants, its role is less clear. Expression of fission yeast CDC25 (Spcdc25) in tobacco results in small cell size, premature flowering and increased shoot morphogenetic capacity in culture. When Arath;WEE1 is over-expressed in Arabidopsis, root apical meristem cell size increases, and morphogenetic capacity of cultured hypocotyls is reduced. However expression of Arath;WEE1 in tobacco plants resulted in precocious flowering and increased shoot morphogenesis of stem explants, and in BY2 cultures cell size was reduced. This phenotype is similar to expression of Spcdc25 and is consistent with a dominant negative effect on WEE1 action. Consistent with this putative mechanism, WEE1 protein levels fell and CDKB levels rose prematurely, coinciding with early mitosis. The phenotype is not due to sense-mediated silencing of WEE1, as overall levels of WEE1 transcript were not reduced in BY2 lines expressing Arath;WEE1. However the pattern of native WEE1 transcript accumulation through the cell cycle was altered by Arath;WEE1 expression, suggesting feedback inhibition of native WEE1 transcription.
- MeSH
- geneticky modifikované rostliny MeSH
- kořeny rostlin genetika metabolismus MeSH
- kultivované buňky MeSH
- květy genetika metabolismus MeSH
- listy rostlin genetika metabolismus MeSH
- mitóza genetika MeSH
- protein-serin-threoninkinasy genetika metabolismus MeSH
- proteiny huseníčku genetika metabolismus MeSH
- regulace genové exprese u rostlin MeSH
- stonky rostlin genetika metabolismus MeSH
- tabák cytologie genetika metabolismus MeSH
- velikost buňky MeSH
- výhonky rostlin genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Microtubules of all eukaryotic cells are formed by α- and β-tubulin heterodimers. In addition to the well known cytoplasmic tubulins, a subpopulation of tubulin can occur in the nucleus. So far, the potential function of nuclear tubulin has remained elusive. In this work, we show that α- and β-tubulins of various organisms contain multiple conserved nuclear export sequences, which are potential targets of the Exportin 1/CRM1 pathway. We demonstrate exemplarily that these NES motifs are sufficient to mediate export of GFP as model cargo and that this export can be inhibited by leptomycin B, an inhibitor of the Exportin 1/CRM1 pathway. Likewise, leptomycin B causes accumulation of GFP-tagged tubulin in interphase nuclei, in both plant and animal model cells. Our analysis of nuclear tubulin content supports the hypothesis that an important function of nuclear tubulin export is the exclusion of tubulin from interphase nuclei, after being trapped by nuclear envelope reassembly during telophase.
- MeSH
- aktivní transport - buněčné jádro fyziologie MeSH
- buněčné jádro metabolismus MeSH
- buněčné linie MeSH
- cytoplazma metabolismus MeSH
- eukaryotické buňky metabolismus MeSH
- karyoferiny metabolismus MeSH
- lidé MeSH
- mikrotubuly metabolismus MeSH
- receptory cytoplazmatické a nukleární metabolismus MeSH
- tabák metabolismus MeSH
- transport proteinů fyziologie MeSH
- tubulin metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Rate of photosynthesis and related plant carbohydrate status are crucial factors affecting plant vigor. Sugars providing carbon and energy sources serve also as important signaling molecules governing plant growth and development through a complex regulatory network. These facts are often neglected when mixotrophic cultivation of plants in vitro is used, where artificial exogenous sugar supply hinders studies of metabolism as well as sugar-driven developmental processes. We compared the growth, selected gas-exchange parameters and sugar metabolism characteristics in four model plants, potato (Solanum tuberosum 'Lada'), tobacco (Nicotiana tabacum 'Samsun'), rapeseed (Brassica napus 'Asgard') and strawberry (Fragaria vesca), under both photomixotrophic (PM) and photoautotrophic (PA) conditions. To ensure PA conditions, we used our improved sun caps that serve as gas and light permeable covers for cultivation vessels. We found bigger biomass accumulation, larger leaf areas, higher stomatal conductance and higher instantaneous water use efficiency and lower root sugar contents in PA plants compared to PM ones. However, for other characteristics (root biomass, root/shoot ratio, pigment contents, leaf sugar and starch levels and transpiration rates), a strong species-dependent reactions to the exogenous sugar supply was noted, which does not allow to create a general view on the overall impact of PM nutrition under in vitro conditions.
- MeSH
- Brassica napus genetika metabolismus fyziologie MeSH
- fotosyntéza genetika fyziologie MeSH
- jahodník genetika metabolismus fyziologie MeSH
- kořeny rostlin genetika metabolismus fyziologie MeSH
- listy rostlin genetika metabolismus fyziologie MeSH
- tabák genetika metabolismus fyziologie MeSH
- Publikační typ
- časopisecké články MeSH
Plum pox virus (PPV, family Potyviridae) is one of the most important viral pathogens of Prunus spp. causing considerable damage to stone-fruit industry worldwide. Among the PPV strains identified so far, only PPV-C, PPV-CR, and PPV-CV are able to infect cherries under natural conditions. Herein, we evaluated the pathogenic potential of two viral isolates in herbaceous host Nicotiana benthamiana. Significantly higher accumulation of PPV capsid protein in tobacco leaves infected with PPV-CR (RU-30sc isolate) was detected in contrast to PPV-C (BY-101 isolate). This result correlated well with the symptoms observed in the infected plants. To further explore the host response upon viral infection at the molecular level, a comprehensive proteomic profiling was performed. Using reverse-phase ultra-high-performance liquid chromatography followed by label-free mass spectrometry quantification, we identified 38 unique plant proteins as significantly altered due to the infection. Notably, the abundances of photosynthesis-related proteins, mainly from the Calvin-Benson cycle, were found more aggressively affected in plants infected with PPV-CR isolate than those of PPV-C. This observation was accompanied by a significant reduction in the amount of photosynthetic pigments extracted from the leaves of PPV-CR infected plants. Shifts in the abundance of proteins that are involved in stimulation of photosynthetic capacity, modification of amino acid, and carbohydrate metabolism may affect plant growth and initiate energy formation via gluconeogenesis in PPV infected N. benthamiana. Furthermore, we suggest that the higher accumulation of H2O2 in PPV-CR infected leaves plays a crucial role in plant defense and development by activating the glutathione synthesis.
- MeSH
- chlorofyl biosyntéza MeSH
- chromatografie s reverzní fází MeSH
- energetický metabolismus genetika MeSH
- fotosyntéza genetika MeSH
- genotyp MeSH
- glutathion biosyntéza MeSH
- hmotnostní spektrometrie MeSH
- interakce hostitele a patogenu genetika MeSH
- karotenoidy biosyntéza MeSH
- listy rostlin genetika metabolismus virologie MeSH
- nemoci rostlin genetika virologie MeSH
- oxidace-redukce MeSH
- peroxid vodíku metabolismus MeSH
- proteiny tepelného šoku klasifikace genetika metabolismus MeSH
- Prunus avium virologie MeSH
- regulace genové exprese u rostlin * MeSH
- rostlinné proteiny klasifikace genetika metabolismus MeSH
- slivoň švestka virologie MeSH
- tabák genetika metabolismus virologie MeSH
- virus šarky švestky klasifikace genetika růst a vývoj patogenita MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem 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
