BACKGROUND: Arsenic toxicity induces a range of metabolic responses in plants, including DNA methylation. The focus of this paper was on the relationship between As-induced stress and plant senescence in the hyperaccumulator Pteris cretica var. Albo-lineata (Pc-Al). We assume difference in physiological parameters and level of DNA methylation in young and old fronds as symptoms of As toxicity. RESULTS: The As accumulation of Pc-Al fronds, grown in pots of haplic chernozem contaminated with 100 mg As kg- 1 for 122 days, decreased with age. Content of As was higher in young than old fronds for variants with 100 mg As kg- 1 (2800 and 2000 mg As kg- 1 dry matter, respectively). The highest As content was determined in old fronds of Pc-Al grown in pots with 250 mg As kg- 1. The increase with age was confirmed for determined nutrients - Cu, Mg, Mn, S and Zn. A significant elevation of all analysed nutrients was showed in old fronds. Arsenic accumulation affected DNA methylation status in fronds, but content of 5-methylcytosine (5mC) decreased only in old fronds of Pc-Al (from 25 to 12%). Determined photosynthetic processes showed a decrease of fluorescence, photosynthetic rate and chlorophylls of As treatments in young and old fronds. Water potential was decreased by As in both fronds. Thinning of the sclerenchymatous inner cortex and a reduction in average tracheid metaxylem in the vascular cylinder was showed in roots of As treatment. Irrespective to fronds age, physiological parameters positively correlated with a 5mC while negatively with direct As toxicity. Opposite results were found for contents of Cu, Mg, Mn, S and Zn. CONCLUSIONS: The results of this paper point to changes in the metabolism of the hyperaccumulator plant Pc-Al, upon low and high exposure to As contamination. The significant impact of As on DNA methylation was found in old fronds. Irrespective to fronds age, significant correlations were confirmed for 5mC and As toxicity. Our analysis of the very low water potential values and lignification of cell walls in roots showed that transports of assimilated metabolites and water between roots and fronds were reduced. As was showed by our results, epigenetic changes could affect studied parameters of the As hyperaccumulator plant Pc-Al, especially in old fronds.
- MeSH
- 5-methylcytosin metabolismus MeSH
- arsen metabolismus toxicita MeSH
- chlorofyl metabolismus MeSH
- ekotoxikologie MeSH
- fotosyntéza účinky léků MeSH
- kořeny rostlin metabolismus MeSH
- látky znečišťující půdu metabolismus MeSH
- listy rostlin metabolismus MeSH
- metylace DNA účinky léků MeSH
- poškození DNA * účinky léků MeSH
- Pteris metabolismus MeSH
- živiny metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
Hebeloma mesophaeum is an ectomycorrhizal fungus frequently associated with metal disturbed environments. In this work, we examined Ag, Cd, and Zn tolerance of H. mesophaeum isolates from heavy metal-polluted (isolate Prib) and clean (isolate Rez) sites. Both mycelia showed essentially the same level of Ag and Zn tolerance, but Prib was more Cd tolerant. In short-term exposures, Prib accumulated slightly less Cd than Rez. Size exclusion chromatography of cell-free extracts and fluorescence microscopy of hyphae with a Cd-specific fluorescent tracer revealed that substantial proportion of Cd was contained in the vacuoles in both isolates. Considering that the proportion of Cd associated with fractions attributable to Cd complexes with cytosolic, metallothionein (MT) peptides was higher in Prib, we examined the copy number and basal levels of HmMTs genes in Rez and Prib. While no difference between the isolates was observed in the gene copy numbers and basal levels of HmMT1 transcripts, the basal transcription of HmMT3 was 3-fold higher in Prib. These observations suggest that MTs provide in Prib better protection against Cd. Furthermore, the higher Cd tolerance in Prib can be to some extent also supported by the efflux or reduced uptake of Cd in the hyphae.
- MeSH
- antifungální látky metabolismus MeSH
- exprese genu * MeSH
- Hebeloma účinky léků izolace a purifikace MeSH
- kadmium toxicita MeSH
- látky znečišťující životní prostředí metabolismus MeSH
- metalothionein biosyntéza genetika MeSH
- mikrobiologie životního prostředí MeSH
- mycelium účinky léků MeSH
- stříbro metabolismus MeSH
- těžké kovy metabolismus MeSH
- tolerance léku * MeSH
- zinek metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
Homeostatic mechanisms preventing the toxicity of heavy metal ions in cells involve, among others, compartmentalization and binding with peptidaceous ligands, particularly the cysteinyl-rich metallothioneins (MTs). We have previously shown that in natural conditions Zn-overaccumulating ectomycorrhizal (EM) fungus Russula bresadolae stores nearly 40% of Zn bound with cysteinyl- and hystidyl-containing RaZBP peptides, which resemble MTs, while the detoxification of Zn and Cd in EM Hebeloma mesophaeum relies upon compartmentalization in small vesicles and vacuoles, respectively. Here, we examined the performance of RaZBP1 gene expressed in H. mesophaeum mycelium with respect to handling of Zn and Cd. Expression of RaZBP1 impaired growth of the mycelium on low-Zn medium by 60%, the growth was partly ameliorated upon the addition of Zn and remained considerable up to 2 mmol/L Zn, while the growth of the wild-type and control mycelia transformed with empty T-DNA was severely reduced in the presence of 0.5 mmol/L Zn; furthermore, RaZBP1 slightly added to Cd tolerance in the range of Cd concentrations of 0.625 to 8 μmol/L. Staining of Zn- or Cd-exposed hyphal cells with Zn- or Cd-specific fluorescent tracers did not indicate that the expression of RaZBP1 would redirect the flow of the metals away from their innate sinks. Size exclusion chromatography of extracted metal species revealed that the complexes corresponding to Zn/Cd-RaZBP1 are present only in minute levels. Considering that RaZBP1 inhibited growth at low Zn, and despite the benefit that it provided to H. mesophaeum in the presence of high Zn and moderate Cd, these data indicate that the binding of excess Zn and Cd with RaZBP1 is not a trait that would be outright transmitted to H. mesophaeum.
- MeSH
- Basidiomycota genetika MeSH
- cytoplazmatické vezikuly metabolismus MeSH
- fungální proteiny genetika metabolismus MeSH
- Hebeloma genetika růst a vývoj metabolismus MeSH
- kadmium metabolismus MeSH
- metalothionein genetika metabolismus MeSH
- mycelium genetika růst a vývoj metabolismus MeSH
- mykorhiza genetika MeSH
- rekombinantní proteiny genetika metabolismus MeSH
- zinek metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
It has been firmly established that macrofungi can accumulate large amounts of heavy metals in their sporocarps. However, the mechanisms of the accumulation and storage are being uncovered only recently. We have previously documented that Russula bresadolae can accumulate over 1 g Zn kg-1 dry weight and that sequestration of a substantial proportion of overaccumulated Zn involves binding with peptides, RaZBPs, seen so far only in this species. In this work we examined Zn contents of 360 sporocarp collections from unpolluted environments covering 114 species of the genus Russula. Whilst the concentrations of Zn in most analysed species were in the range of 50-150 mg kg-1, the species of subgenera Brevipes and Compactae accumulate very low Zn (< 50 mg kg-1). We further identified five new Zn-overaccumulating species of subgenus Russula, which form with R. bresadolae a separate phylogenetic subclade in which the sporocarp Zn concentrations ranged from 326 to 845 mg kg-1. We demonstrate that R. pumila and R. ochroleuca express at least one ZBP gene and when expressed in metal-sensitive S. cerevisiae, all ZBPs protected the yeasts against Zn (and Cd) toxicity equally well. The respective ZBPs were confirmed in the native Zn-complexes of R. pumila and R. ochroleuca, which represented 80% of Zn extracted from their sporocarps. This study is the first extensive genus-wide report of metal accumulation in macrofungi, which further demonstrates that the Zn binding with cytosolic ZBP peptides is not a trait restricted only to R. bresadolae.
A search of R. atropurpurea transcriptome for sequences encoding the transporters of the Zrt-, Irt-like Protein (ZIP) family, which are in eukaryotes integral to Zn supply into cytoplasm, allowed the identification of RaZIP1 cDNA with a predicted product belonging to ZIP I subfamily; it was subjected to functional studies in mutant Saccharomyces cerevisiae strains. The expression of RaZIP1, but not RaZIP1H208A or RaZIP1H232A mutants lacking conserved-among-ZIPs transmembrane histidyls, complemented Zn uptake deficiency in zrt1Δzrt2Δ yeasts. RaZIP1 substantially increased cellular Zn uptake in this strain and added to Zn sensitivity in zrc1Δcot1Δ mutant. The Fe uptake deficiency in ftr1Δ strain was not rescued and Mn uptake was insufficient for toxicity in Mn-sensitive pmr1Δ yeasts. By contrast, RaZIP1 increased Cd sensitivity in yap1Δ strain and conferred Cd transport activity in yeasts, albeit with substantially lower efficiency compared to Zn transport. In metal uptake assays, the accumulation of Zn in zrt1Δzrt2Δ strain remained unaffected by Cd, Fe, and Mn present in 20-fold molar excess over Zn. Immunofluorescence microscopy detected functional hemagglutinin-tagged HA::RaZIP1 on the yeast cell protoplast periphery. Altogether, these data indicate that RaZIP1 is a high-affinity plasma membrane transporter specialized in Zn uptake, and improve the understanding of the cellular and molecular biology of Zn in R. atropurpurea that is known for its ability to accumulate remarkably high concentrations of Zn.
- MeSH
- Basidiomycota genetika růst a vývoj MeSH
- biologický transport účinky léků MeSH
- fungální proteiny genetika metabolismus MeSH
- mykorhiza chemie genetika MeSH
- proteiny přenášející kationty MeSH
- regulace genové exprese u rostlin MeSH
- rostlinné proteiny chemie genetika metabolismus MeSH
- sekvence aminokyselin genetika MeSH
- zinek chemie metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Beer production is one of the oldest known traditional biotechnological processes, but is nowadays facing increasing demands not only for enhanced product quality, but also for improved production economics. Targeted genetic modification of a yeast strain is one way to increase beer quality and to improve the economics of beer production. In this review we will present current knowledge on traditional approaches for improving brewing strains and for rational metabolic engineering. These research efforts will, in the near future, lead to the development of a wider range of industrial strains that should increase the diversity of commercial beers.
- MeSH
- CRISPR-Cas systémy MeSH
- fermentace MeSH
- genetické inženýrství metody MeSH
- geneticky modifikované mikroorganismy genetika fyziologie MeSH
- genom fungální MeSH
- lidé MeSH
- metabolické inženýrství metody MeSH
- pivo * MeSH
- plazmidy genetika MeSH
- regulace genové exprese u hub MeSH
- Saccharomyces cerevisiae genetika fyziologie MeSH
- veřejné mínění MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Compared to most other alcoholic beverages, the shelf life of beer is much more limited due to its instability in the bottle. That instability is most likely to appear as turbidity (haze), even sedimentation, during storage. The haze in beer is mostly caused by colloidal particles formed by interactions between proteins and polyphenols within the beer. Therefore, beers are usually stabilized by removing at least one of these components. We developed and constructed a Saccharomyces cerevisiae strain with a proline-rich QPF peptide attached to the cell wall, using the C-terminal anchoring domain of α-agglutinin. The QPF peptide served to bind polyphenols during fermentation and, thus, to decrease their concentration. Strains displaying QPF were able to bind about twice as much catechin and epicatechin as a control strain displaying only the anchoring domain. All these experiments were done with model solutions. Depending on the concentration of yeast, uptake of polyphenols was 1.7-2.5 times higher. Similarly, the uptake of proanthocyanidins was increased by about 20 %. Since the modification of yeasts with QPF did not affect their fermentation performance under laboratory conditions, the display of QPF appears to be an approach to increase the stability of beer.
Mechanisms evolved in eukaryotes to handle heavy metals involve cytosolic, metal-binding metallothioneins (MTs). We have previously documented that the sequestration of silver (Ag) in the Ag-hyperaccumulating Amanita strobiliformis is dominated by 34-amino-acid (AA) AsMT1a, 1b, and 1c isoforms. Here we show that in addition to AsMT1a, 1b, and 1c isogenes, the fungus has two other MT genes: AsMT2 encoding a 34-AA AsMT2 similar to MTs known from other species, but unrelated to AsMT1s; AsMT3 coding for a 62-AA AsMT3 that shares substantial identity with as-yet-uncharacterized conserved peptides predicted in agaricomycetes. Transcription of AsMT1s and AsMT3 in the A. strobiliformis mycelium was specifically inducible by treatments with Ag or copper (Cu) and zinc (Zn) or cadmium (Cd), respectively; AsMT2 showed a moderate upregulation in the presence of Cd. Expression of AsMTs in the metal-sensitive Saccharomyces cerevisiae revealed that all AsMTs confer increased Cd tolerance (AsMT3 proved the most effective) and that, unlike AsMT1 and AsMT2, AsMT3 can protect the yeasts against Zn toxicity. The highest level of Cu tolerance was observed with yeasts expressing AsMT1a. Our data indicate that A. strobiliformis can specifically employ different MT genes for functions in the cellular handling of Ag and Cu (AsMT1s) and Zn (AsMT3).
- MeSH
- Amanita genetika metabolismus MeSH
- genetická transkripce účinky léků MeSH
- měď metabolismus MeSH
- metalothionein genetika metabolismus MeSH
- Saccharomyces cerevisiae účinky léků genetika metabolismus MeSH
- stanovení celkové genové exprese MeSH
- stříbro metabolismus MeSH
- tolerance léku MeSH
- zinek metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
OBJECTIVES: To convert α-acetolactate into acetoin by an α-acetolactate decarboxylase (ALDC) to prevent its conversion into diacetyl that gives beer an unfavourable buttery flavour. RESULTS: We constructed a whole Saccharomyces cerevisiae cell catalyst with a truncated active ALDC from Acetobacter aceti ssp xylinum attached to the cell wall using the C-terminal anchoring domain of α-agglutinin. ALDC variants in which 43 and 69 N-terminal residues were absent performed equally well and had significantly decreased amounts of diacetyl during fermentation. With these cells, the highest concentrations of diacetyl observed during fermentation were 30 % less than those in wort fermented with control yeasts displaying only the anchoring domain and, unlike the control, virtually no diacetyl was present in wort after 7 days of fermentation. CONCLUSIONS: Since modification of yeasts with ALDC variants did not affect their fermentation performance, the display of α-acetolactate decarboxylase activity is an effective approach to decrease the formation of diacetyl during beer fermentation.
Ectomycorrhizal (ECM) fungi contribute to the survival of host trees on metal-rich soils by reducing the transfer of toxic metals into roots. However, little is known about the ability of ECM fungi to accumulate elements in ectomycorrhizae (ECMs). Here we report Ag, As, Cd, Cl, Cu, Sb, V, and Zn contents in wild-grown Norway spruce ECMs collected in a smelter-polluted area at Lhota near Příbram, Czech Republic. The ECMs data were compared with the element concentrations determined in the corresponding non-mycorrhizal fine roots, soils, and soil extracts. Bioaccumulation factors were calculated to differentiate the element accumulation ability of ECMs inhabited by different mycobionts, which were identified by ITS rDNA sequencing. Among the target elements, the highest contents were observed for Ag, Cl, Cd, and Zn; Imleria badia ECMs showed the highest capability to accumulate these elements. ECMs of Amanita muscaria, but not of other species, accumulated V. The analysis of the proportions of I. badia and A. muscaria mycelia in ECMs by using species-specific quantitative real-time PCR revealed variable extent of the colonization of roots, with median values close to 5% (w/w). Calculated Ag, Cd, Zn and Cl concentrations in the mycelium of I. badia ECMs were 1 680, 1 510, 2 670, and 37,100 mg kg(-1) dry weight, respectively, indicating substantial element accumulation capacity of hyphae of this species in ECMs. Our data strengthen the idea of an active role of ECM fungi in soil-fungal-plant interactions in polluted environments.
- MeSH
- chlor metabolismus MeSH
- kořeny rostlin metabolismus MeSH
- látky znečišťující půdu metabolismus MeSH
- mycelium metabolismus MeSH
- mykorhiza metabolismus MeSH
- polokovy metabolismus MeSH
- těžké kovy metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Česká republika MeSH
