Pollen germination as a crucial process in plant development strongly depends on the accessibility of carbon as energy source. Carbohydrates, however, function not only as a primary energy source, but also as important signaling components. In a comprehensive study, we analyzed various aspects of the impact of 32 different sugars on in vitro germination of Arabidopsis pollen comprising about 150 variations of individual sugars and combinations. Twenty-six structurally different mono-, di- and oligosaccharides, and sugar analogs were initially tested for their ability to support pollen germination. Whereas several di- and oligosaccharides supported pollen germination, hexoses such as glucose, fructose and mannose did not support and even considerably inhibited pollen germination when added to germination-supporting medium. Complementary experiments using glucose analogs with varying functional features, the hexokinase inhibitor mannoheptulose and the glucose-insensitive hexokinase-deficient Arabidopsis mutant gin2-1 suggested that mannose- and glucose-mediated inhibition of sucrose-supported pollen germination depends partially on hexokinase signaling. The results suggest that, in addition to their role as energy source, sugars act as signaling molecules differentially regulating the complex process of pollen germination depending on their structural properties. Thus, a sugar-dependent multilayer regulation of Arabidopsis pollen germination is supported, which makes this approach a valuable experimental system for future studies addressing sugar sensing and signaling.

• MeSH
• Arabidopsis účinky léků   fyziologie   MeSH
• hexosy metabolismus   farmakologie   MeSH
• klíčení účinky léků   fyziologie   MeSH
• mannosa metabolismus   farmakologie   MeSH
• metabolismus sacharidů * MeSH
• oligosacharidy chemie   metabolismus   farmakologie   MeSH
• pyl metabolismus   fyziologie   MeSH
• sacharidy MeSH
• sacharosa metabolismus   farmakologie   MeSH
• Publikační typ
• časopisecké články MeSH

Polarized exocytosis is critical for pollen tube growth, but its localization and function are still under debate. The exocyst vesicle-tethering complex functions in polarized exocytosis. Here, we show that a sec3a exocyst subunit null mutant cannot be transmitted through the male gametophyte due to a defect in pollen tube growth. The green fluorescent protein (GFP)-SEC3a fusion protein is functional and accumulates at or proximal to the pollen tube tip plasma membrane. Partial complementation of sec3a resulted in the development of pollen with multiple tips, indicating that SEC3 is required to determine the site of pollen germination pore formation. Time-lapse imaging demonstrated that SEC3a and SEC8 were highly dynamic and that SEC3a localization on the apical plasma membrane predicts the direction of growth. At the tip, polar SEC3a domains coincided with cell wall deposition. Labeling of GFP-SEC3a-expressing pollen with the endocytic marker FM4-64 revealed the presence of subdomains on the apical membrane characterized by extensive exocytosis. In steady-state growing tobacco (Nicotiana tabacum) pollen tubes, SEC3a displayed amino-terminal Pleckstrin homology-like domain (SEC3a-N)-dependent subapical membrane localization. In agreement, SEC3a-N interacted with phosphoinositides in vitro and colocalized with a phosphatidylinositol 4,5-bisphosphate (PIP2) marker in pollen tubes. Correspondingly, molecular dynamics simulations indicated that SEC3a-N associates with the membrane by interacting with PIP2 However, the interaction with PIP2 is not required for polar localization and the function of SEC3a in Arabidopsis (Arabidopsis thaliana). Taken together, our findings indicate that SEC3a is a critical determinant of polar exocytosis during tip growth and suggest differential regulation of the exocytotic machinery depending on pollen tube growth modes.

• MeSH
• Arabidopsis genetika   růst a vývoj   metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• časosběrné zobrazování metody   MeSH
• exocytóza * MeSH
• fosfatidylinositol-4,5-difosfát metabolismus   MeSH
• fosfatidylinositoly metabolismus   MeSH
• fylogeneze MeSH
• geneticky modifikované rostliny MeSH
• konfokální mikroskopie MeSH
• mutace MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• protein - isoformy genetika   metabolismus   MeSH
• proteiny huseníčku klasifikace   genetika   metabolismus   MeSH
• pyl genetika   růst a vývoj   metabolismus   MeSH
• pylová láčka genetika   růst a vývoj   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• sekvenční homologie aminokyselin MeSH
• sekvenční homologie nukleových kyselin MeSH
• simulace molekulární dynamiky MeSH
• stanovení celkové genové exprese metody   MeSH
• vazba proteinů MeSH
• vazebná místa genetika   MeSH
• vezikulární transportní proteiny klasifikace   genetika   metabolismus   MeSH
• zelené fluorescenční proteiny genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH

Allergy to the short ragweed (Ambrosia artemisiifolia) pollen is a major health problem. The ragweed allergen repertoire has been recently expanded with the identification of Amb a 11, a new major allergen belonging to the cysteine protease family. To better characterize Amb a 11, a recombinant proform of the molecule with a preserved active site was produced in Escherichia coli, refolded, and processed in vitro into a mature enzyme. The enzymatic activity is revealed by maturation following an autocatalytic processing resulting in the cleavage of both N- and C-terminal propeptides. The 2.05-Å resolution crystal structure of pro-Amb a 11 shows an overall typical C1A cysteine protease fold with a network of molecular interactions between the N-terminal propeptide and the catalytic triad of the enzyme. The allergenicity of Amb a 11 was confirmed in a murine sensitization model, resulting in airway inflammation, production of serum IgEs, and induction of Th2 immune responses. Of note, inflammatory responses were higher with the mature form, demonstrating that the cysteine protease activity critically contributes to the allergenicity of the molecule. Collectively, our results clearly demonstrate that Amb a 11 is a bona fide cysteine protease exhibiting a strong allergenicity. As such, it should be considered as an important molecule for diagnosis and immunotherapy of ragweed pollen allergy.

• MeSH
• alergeny chemie   imunologie   MeSH
• antigeny rostlinné imunologie   MeSH
• cysteinové proteasy chemie   imunologie   MeSH
• katalytická doména MeSH
• konzervovaná sekvence MeSH
• krystalografie rentgenová MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• myši inbrední BALB C MeSH
• posttranslační úpravy proteinů MeSH
• prekurzory enzymů chemie   imunologie   MeSH
• proteolýza MeSH
• rostlinné extrakty imunologie   MeSH
• rostlinné proteiny chemie   imunologie   MeSH
• sekvence aminokyselin MeSH
• sezónní alergická rýma imunologie   prevence a kontrola   MeSH
• vodíková vazba MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The vesicle-tethering complex exocyst is one of the crucial cell polarity regulators. The EXO70 subunit is required for the targeting of the complex and is represented by many isoforms in angiosperm plant cells. This diversity could be partly responsible for the establishment and maintenance of membrane domains with different composition. To address this hypothesis, we employed the growing pollen tube, a well-established cell polarity model system, and performed large-scale expression, localization, and functional analysis of tobacco (Nicotiana tabacum) EXO70 isoforms. Various isoforms localized to different regions of the pollen tube plasma membrane, apical vesicle-rich inverted cone region, nucleus, and cytoplasm. The overexpression of major pollen-expressed EXO70 isoforms resulted in growth arrest and characteristic phenotypic deviations of tip swelling and apical invaginations. NtEXO70A1a and NtEXO70B1 occupied two distinct and mutually exclusive plasma membrane domains. Both isoforms partly colocalized with the exocyst subunit NtSEC3a at the plasma membrane, possibly forming different exocyst complex subpopulations. NtEXO70A1a localized to the small area previously characterized as the site of exocytosis in the tobacco pollen tube, while NtEXO70B1 surprisingly colocalized with the zone of clathrin-mediated endocytosis. Both NtEXO70A1a and NtEXO70B1 colocalized to different degrees with markers for the anionic signaling phospholipids phosphatidylinositol 4,5-bisphosphate and phosphatidic acid. In contrast, members of the EXO70 C class, which are specifically expressed in tip-growing cells, exhibited exocytosis-related functional effects in pollen tubes despite the absence of apparent plasma membrane localization. Taken together, our data support the existence of multiple membrane-trafficking domains regulated by different EXO70-containing exocyst complexes within a single cell.

• MeSH
• buněčná membrána metabolismus   MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• exocytóza genetika   MeSH
• fylogeneze MeSH
• hmotnostní spektrometrie s elektrosprejovou ionizací MeSH
• konfokální mikroskopie MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• protein - isoformy genetika   metabolismus   MeSH
• proteomika metody   MeSH
• pylová láčka genetika   růst a vývoj   metabolismus   MeSH
• regulace genové exprese u rostlin MeSH
• rostlinné proteiny klasifikace   genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• sekvenční homologie aminokyselin MeSH
• tabák genetika   metabolismus   MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• vývojová regulace genové exprese MeSH
• Publikační typ
• časopisecké články MeSH

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.

• MeSH
• 2D gelová elektroforéza MeSH
• chromatografie afinitní metody   MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• fosfoproteiny analýza   chemie   izolace a purifikace   MeSH
• fosforylace MeSH
• molekulární sekvence - údaje MeSH
• proteom analýza   chemie   MeSH
• pyl chemie   MeSH
• rostlinné proteiny analýza   chemie   MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• tabák chemie   MeSH
• titan MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The way pollinators gather resources may play a key role for buffering their population declines. Social pollinators like bumblebees could adjust their foraging after significant workforce reductions to keep provisions to the colony optimal, especially in terms of pollen diversity and quantity. To test what effects a workforce reduction causes on the foraging for pollen, commercially-acquired colonies of the bumblebee Bombus terrestris were allowed to forage in the field and they were experimentally manipulated by removing half the number of workers. For each bumblebee, the pollen pellets were taxonomically identified with DNA metabarcoding of the ITS2 region followed by a statistical filtering based on ROC curves to filter out underrepresented OTUs. Video cameras and network analyses were employed to investigate changes in foraging strategies and behaviour. After filtering out the false-positives, HTS metabarcoding yielded a high plant diversity in the pollen pellets; for plant identity and pollen quantity traits no differences emerged between samples from treated and from control colonies, suggesting that plant choice was influenced mainly by external factors such as the plant phenology. The colonies responded to the removal of 50% of their workers by increasing the foraging activity of the remaining workers, while only negligible changes were found in diet breadth and indices describing the structure of the pollen transport network. Therefore, a consistency in the bumblebees' feeding strategies emerges in the short term despite the lowered workforce.

• MeSH
• biodiverzita MeSH
• fyziologie výživy zvířat MeSH
• opylení fyziologie   MeSH
• populační dynamika MeSH
• pyl * genetika   MeSH
• rostliny klasifikace   genetika   MeSH
• stravovací zvyklosti MeSH
• taxonomické DNA čárové kódování MeSH
• včely fyziologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika MeSH

Heat shock transcription factors (Hsfs) are involved in multiple aspects of stress response and plant growth. However, their role during male gametophyte development is largely unknown, although the generative phase is the most sensitive and critical period in the plant life cycle. Based on a wide screen of T-DNA mutant lines, we identified the atren1 mutation (restricted to nucleolus1) in early male gametophytic gene At1g77570, which has the closest homology to HSFA5 gene, the member of a heat shock transcription factor (HSF) gene family. The mutation causes multiple defects in male gametophyte development in both structure and function. Because the mutation disrupts an early acting (AtREN1) gene, these pollen phenotype abnormalities appear from bicellular pollen stage to pollen maturation. Moreover, the consequent progamic phase is compromised as well as documented by pollen germination defects and limited transmission via male gametophyte. In addition, atren1/- plants are defective in heat stress (HS) response and produce notably higher proportion of aberrant pollen grains. AtREN1 protein is targeted specifically to the nucleolus that, together with the increased size of the nucleolus in atren1 pollen, suggests that it is likely to be involved in ribosomal RNA biogenesis or other nucleolar functions.

• MeSH
• alely MeSH
• Arabidopsis cytologie   růst a vývoj   metabolismus   MeSH
• buněčné jadérko metabolismus   MeSH
• DNA bakterií genetika   MeSH
• DNA vazebné proteiny genetika   metabolismus   MeSH
• exony genetika   MeSH
• fenotyp MeSH
• klíčení MeSH
• mutace genetika   MeSH
• penetrance MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• pyl cytologie   genetika   růst a vývoj   MeSH
• pylová láčka cytologie   genetika   růst a vývoj   MeSH
• reakce na tepelný šok * genetika   MeSH
• regulace genové exprese u rostlin MeSH
• segregace chromozomů genetika   MeSH
• testy genetické komplementace MeSH
• transport proteinů MeSH
• vývojová regulace genové exprese MeSH
• zelené fluorescenční proteiny metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• alergeny chemie   imunologie   MeSH
• antigeny rostlinné chemie   imunologie   MeSH
• gibereliny chemie   imunologie   MeSH
• hmotnostní spektrometrie MeSH
• imunoglobulin E imunologie   MeSH
• lidé MeSH
• potravinová alergie diagnóza   imunologie   MeSH
• pyl chemie   imunologie   MeSH
• rostlinné proteiny chemie   imunologie   MeSH
• sekvence aminokyselin MeSH
• zkřížené reakce imunologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• dopisy MeSH
• práce podpořená grantem MeSH

Precise guided pollen tube growth by the female gametophyte is a prerequisite for successful sexual reproduction in flowering plants. Cysteine-rich proteins (CRPs) secreted from the embryo sac are known pollen tube attractants perceived by pollen tube receptor-like kinases. How pre-mRNA splicing facilitates this cell-to-cell communication is not understood. Here, we report a novel function of Pre-mRNA PROCESSING factor 8 paralogs, PRP8A and PRP8B, as regulators of pollen tube attraction. Double mutant prp8a prp8b ovules cannot attract pollen tubes, and prp8a prp8b pollen tubes fail to sense the ovule's attraction signals. Only 3% of ovule-expressed genes were misregulated in prp8a prp8b Combination of RNA sequencing and the MYB98/LURE1.2-YFP reporter revealed that the expression of MYB98, LUREs and 49 other CRPs were downregulated, suggesting loss of synergid cell fate. Differential exon usage and intron retention analysis revealed autoregulation of PPR8A/PRP8B splicing. In vivo, PRP8A co-immunoprecipitates with splicing enhancer AtSF3A1, suggesting involvement of PRP8A in 3'-splice site selection. Our data hint that the PRP8A/PRP8B module exhibits spliceosome autoregulation to facilitate pollen tube attraction via transcriptional regulation of MYB98, CRPs and LURE pollen tube attractants.

• MeSH
• Arabidopsis metabolismus   MeSH
• fluorescenční mikroskopie MeSH
• geneticky modifikované rostliny metabolismus   MeSH
• místa sestřihu RNA MeSH
• mutageneze MeSH
• podjednotky proteinů genetika   metabolismus   MeSH
• proteiny huseníčku chemie   genetika   metabolismus   MeSH
• proteiny vázající RNA chemie   genetika   metabolismus   MeSH
• pylová láčka růst a vývoj   metabolismus   MeSH
• regulace genové exprese u rostlin MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• sestřihové faktory genetika   metabolismus   MeSH
• spliceozomy metabolismus   MeSH
• transkripční faktory genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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.

• MeSH
• Arabidopsis metabolismus   MeSH
• fosfoproteiny metabolismus   MeSH
• proteom metabolismus   MeSH
• pyl metabolismus   MeSH
• rostlinné proteiny metabolismus   MeSH
• tabák metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH