We investigated feasibility of the Full-length complementary DNA OvereXpression (FOX) system as a mutagenesis approach in poplar, using developing xylem tissue. The main goal was to assess the overall mutation rate and if the system will increase instances of mutants affected in traits linked to the xylem tissue. Indeed, we found a high mutation rate of 17.7%, whereas 80% of all mutants were significantly affected in cellulose, lignin and/or hemicellulose. Cell wall biosynthesis is a major process occurring during xylem development. Enrichment of mutants affected in cell wall composition suggests that the tissue source for the FOX library influenced the occurrence of mutants affected in a trait linked to this tissue. Additionally, we found that FLcDNAs from mutants affected in cell wall composition were homologous to genes known to be involved in cell wall biosynthesis and most recovered FLcDNAs corresponded to genes whose native expression was highest in xylem. We characterized in detail a mutant line with increased diameter. The phenotype was caused by a poplar homolog of LONELY GUY 1 (LOG1), which encodes an enzyme in cytokinin biosynthesis and significantly increased xylem proliferation. The causative role of LOG1 in the observed phenotype was further reaffirmed by elevated cytokinin concentration in the mutant and recapitulation overexpression experiment wherein multiple independent lines phenocopied the original FOX mutant. Our experiments show that the FOX approach can be efficiently used for gene discovery and molecular interrogation of traits specific to woody perennial growth and development.
- MeSH
- buněčná stěna genetika metabolismus MeSH
- dřevo * genetika metabolismus MeSH
- genová knihovna MeSH
- lignin metabolismus MeSH
- mutační rychlost MeSH
- Populus * genetika metabolismus MeSH
- regulace genové exprese u rostlin genetika MeSH
- rostlinné proteiny genetika metabolismus MeSH
- xylém genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
Element content and expression of genes of interest on single cell types, such as stomata, provide valuable insights into their specific physiology, improving our understanding of leaf gas exchange regulation. We investigated how far differences in stomatal conductance (gs ) can be ascribed to changes in guard cells functioning in amphistomateous leaves. gs was measured during the day on both leaf sides, on well-watered and drought-stressed trees (two Populus euramericana Moench and two Populus nigra L. genotypes). In parallel, guard cells were dissected for element content and gene expressions analyses. Both were strongly arranged according to genotype, and drought had the lowest impact overall. Normalizing the data by genotype highlighted a structure on the basis of leaf sides and time of day both for element content and gene expression. Guard cells magnesium, phosphorus, and chlorine were the most abundant on the abaxial side in the morning, where gs was at the highest. In contrast, genes encoding H+ -ATPase and aquaporins were usually more abundant in the afternoon, whereas genes encoding Ca2+ -vacuolar antiporters, K+ channels, and ABA-related genes were in general more abundant on the adaxial side. Our work highlights the unique physiology of each leaf side and their analogous rhythmicity through the day.
- MeSH
- genotyp MeSH
- komplementární DNA genetika izolace a purifikace MeSH
- listy rostlin genetika metabolismus MeSH
- mikroanalýza elektronovou sondou MeSH
- období sucha MeSH
- Populus klasifikace genetika metabolismus MeSH
- protonové ATPasy genetika metabolismus MeSH
- průduchy rostlin genetika metabolismus MeSH
- regulace genové exprese u rostlin MeSH
- RNA rostlin genetika izolace a purifikace MeSH
- rostlinné proteiny genetika metabolismus MeSH
- stromy genetika metabolismus MeSH
- transpirace rostlin fyziologie MeSH
- voda fyziologie MeSH
- vývoj rostlin MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
MAIN CONCLUSION: Five poplar CHASE-containing histidine kinase receptors bind cytokinins and display kinase activities. Both endogenous isoprenoid and aromatic cytokinins bind to the receptors in live cell assays. Cytokinins are phytohormones that play key roles in various developmental processes in plants. The poplar species Populus × canadensis, cv. Robusta, is the first organism found to contain aromatic cytokinins. Here, we report the functional characterization of five CHASE-containing histidine kinases from P. × canadensis: PcHK2, PcHK3a, PcHK3b, PcHK4a and PcHK4b. A qPCR analysis revealed high transcript levels of all PcHKs other than PcHK4b across multiple poplar organs. The ligand specificity was determined using a live cell Escherichia coli assay and we provide evidence based on UHPLC-MS/MS data that ribosides can be true ligands. PcHK2 exhibited higher sensitivity to iP-type cytokinins than the other receptors, while PcHK3a and PcHK3b bound these cytokinins much more weakly, because they possess two isoleucine residues that clash with the cytokinin base and destabilize its binding. All receptors display kinase activity but their activation ratios in the presence/absence of cytokinin differ significantly. PcHK4a displays over 400-fold higher kinase activity in the presence of cytokinin, suggesting involvement in strong responses to changes in cytokinin levels. trans-Zeatin was both the most abundant cytokinin in poplar and that with the highest variation in abundance, which is consistent with its strong binding to all five HKs and activation of cytokinin signaling via A-type response regulators. The aromatic cytokinins' biological significance remains unclear, their levels vary diurnally, seasonally, and annually. PcHK3 and PcHK4 display the strongest binding at pH 7.5 and 5.5, respectively, in line with their putative membrane localization in the endoplasmic reticulum and plasma membrane.
MAIN CONCLUSION: Isoprenoid and aromatic cytokinins occur in poplar as free compounds and constituents of tRNA, poplar isopentenyltransferases are involved in the production of isoprenoid cytokinins, while biosynthesis of their aromatic counterparts remains unsolved. Cytokinins are phytohormones with a fundamental role in the regulation of plant growth and development. They occur naturally either as isoprenoid or aromatic derivatives, but the latter are quite rare and less studied. Here, the spatial expression of all nine isopentenyl transferase genes of Populus × canadensis cv. Robusta (PcIPTs) as analyzed by RT-qPCR revealed a tissue preference and strong differences in expression levels for the different adenylate and tRNA PcIPTs. Together with their phylogeny, this result suggests a functional diversification for the different PcIPT proteins. Additionally, the majority of PcIPT genes were cloned and expressed in Arabidopsis thaliana under an inducible promoter. The cytokinin levels measured in the Arabidopsis-overexpressing lines as well as their phenotype indicate that the studied adenylate and tRNA PcIPT proteins are functional in vivo and thus will contribute to the cytokinin pool in poplar. We screened the cytokinin content in leaves of 12 Populus species by ultra-high performance-tandem mass spectrometry (UHPLC-MS/MS) and discovered that the capacity to produce not only isoprenoid, but also aromatic cytokinins is widespread amongst the Populus accessions studied. Important for future studies is that the levels of aromatic cytokinins transiently increase after daybreak and are much higher in older plants.
- MeSH
- alkyltransferasy a aryltransferasy genetika metabolismus MeSH
- Arabidopsis genetika metabolismus MeSH
- cytokininy biosyntéza MeSH
- fylogeneze MeSH
- geneticky modifikované rostliny MeSH
- listy rostlin genetika metabolismus MeSH
- Populus genetika metabolismus MeSH
- regulátory růstu rostlin metabolismus MeSH
- rostlinné proteiny genetika metabolismus MeSH
- tandemová hmotnostní spektrometrie MeSH
- Publikační typ
- časopisecké články MeSH
Cytokinins are plant hormones that typically block or delay leaf senescence. We profiled 34 different cytokinins/cytokinin metabolites (including precursors, conjugates and degradation products) in leaves of a free-growing mature aspen (Populus tremula) before and after the initiation of autumnal senescence over three consecutive years. The levels and profiles of individual cytokinin species, or classes/groups, varied greatly between years, despite the fact that the onset of autumn senescence was at the same time each year, and senescence was not associated with depletion of either active or total cytokinin levels. Levels of aromatic cytokinins (topolins) were low and changed little over the autumn period. Diurnal variations and weather-dependent variations in cytokinin content were relatively limited. We also followed the expression patterns of all aspen genes implicated as having roles in cytokinin metabolism or signalling, but neither the pattern of regulation of any group of genes nor the expression of any particular gene supported the notion that decreased cytokinin signalling could explain the onset of senescence. Based on the results from this tree, we therefore suggest that cytokinin depletion is unlikely to explain the onset of autumn leaf senescence in aspen.
- MeSH
- biosyntetické dráhy genetika MeSH
- chlorofyl metabolismus MeSH
- cytokininy metabolismus MeSH
- down regulace genetika MeSH
- glukosidy metabolismus MeSH
- listy rostlin metabolismus fyziologie MeSH
- počasí MeSH
- Populus genetika metabolismus fyziologie MeSH
- regulace genové exprese u rostlin MeSH
- roční období MeSH
- rostlinné geny MeSH
- stanovení celkové genové exprese MeSH
- Publikační typ
- časopisecké články MeSH
Fast-growing clones of Salix and Populus have been studied for remediation of soils contaminated by risk elements (RE) using short-rotation coppice plantations. Our aim was to assess biomass yield and distributions of elements in wood and bark of highly productive willow (S1--[Salix schwerinii × Salix viminalis] × S. viminalis, S2--Salix × smithiana clone S-218) and poplar (P1--Populus maximowiczii × Populus nigra, P2--P. nigra) clones with respect to aging. The field experiment was established in April 2008 on moderately Cd-, Pb- and Zn- contaminated soil. Shoots were harvested after four seasons (February 2012) and separated into annual classes of wood and bark. All tested clones grew on contaminated soils, with highest biomass production and lowest mortality exhibited by P1 and S2. Concentrations of elements, with exception of Ca and Pb, decreased with age and were higher in bark than in wood. The Salix clones were characterised by higher removal of Cd, Mn and Zn compared to the Populus clones. Despite generally higher RE content in young shoots, partly due to lower wood/bark ratios and higher RE concentrations in bark, the overall removal of RE was higher in older wood classes due to higher biomass yield. Thus, longer rotations seem to be more effective when phytoextraction strategy is considered. Of the four selected clones, S1 exhibited the best removal of Cd and Zn and is a good candidate for phytoextraction.
- MeSH
- biodegradace MeSH
- biomasa MeSH
- dřevo metabolismus MeSH
- kůra rostlin metabolismus MeSH
- látky znečišťující půdu metabolismus MeSH
- Populus metabolismus MeSH
- prvky MeSH
- regenerace a remediace životního prostředí * MeSH
- Salix metabolismus MeSH
- těžké kovy metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
To characterize the phytoextraction efficiency of two clones of willow trees (Salix x smithiana Willd., Salix rubens) and two clones of poplar trees (Populus nigra x maximowiczii, Populus nigra Wolterson) were planted in contaminated soil (0.4-2.0 mg Cd.kg(-1), 78-313 mg Zn.kg(-1), 21.3-118 mg Cu.kg(-1)). Field experiment was carried out in Czech Republic. The study investigated their ability to accumulate heavy metals (Cd, Zn, and Cu) in harvestable plant parts. The poplars produced higher amount of biomass than willows. Both Salix clones accumulated higher amount of Cd, Zn and Cu in their biomass (maximum 6.8 mg Cd.kg(-1), 909 mg Zn.kg(-1), and 17.7 mg Cu.kg(-1)) compared to Populus clones (maximum 2.06 mg Cd.kg(-1), 463 mg Zn.kg(-1), and 11.8 mg Cu.kg(-1)). There were no significant differences between clones of individual species. BCs for Cd and Zn were greater than 1 (the highest in willow leaves). BCs values of Cu were very low. These results indicate that Salix is more suitable plant for phytoextraction of Cd and Zn than Populus. The Cu phytoextraction potential of Salix and Populus trees was not confirmed in this experiment due to low soil availability of this element.
- MeSH
- biodegradace MeSH
- kadmium analýza metabolismus MeSH
- látky znečišťující půdu analýza metabolismus MeSH
- měď analýza metabolismus MeSH
- Populus chemie metabolismus MeSH
- regenerace a remediace životního prostředí přístrojové vybavení metody MeSH
- Salix chemie metabolismus MeSH
- stromy chemie metabolismus MeSH
- zinek analýza metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
Here, we describe a method for the combined metabolomic, proteomic, transcriptomic and genomic analysis from one single sample as a major step for multilevel data integration strategies in systems biology. While extracting proteins and DNA, this protocol also allows the separation of metabolites into polar and lipid fractions, as well as RNA fractionation into long and small RNAs, thus allowing a broad range of transcriptional studies. The isolated biomolecules are suitable for analysis with different methods that range from electrophoresis and blotting to state-of-the-art procedures based on mass spectrometry (accurate metabolite profiling, shot-gun proteomics) or massive sequencing technologies (transcript analysis). The low amount of starting tissue, its cost-efficiency compared with the utilization of commercial kits, and its performance over a wide range of plant, microbial, and algal species such as Chlamydomonas, Arabidopsis, Populus, or Pinus, makes this method a universal alternative for multiple molecular isolation from plant tissues.
- MeSH
- Arabidopsis genetika metabolismus MeSH
- borovice genetika metabolismus MeSH
- Chlamydomonas reinhardtii genetika metabolismus MeSH
- DNA rostlinná izolace a purifikace MeSH
- genomika metody MeSH
- metabolomika metody MeSH
- Populus genetika metabolismus MeSH
- proteomika metody MeSH
- reprodukovatelnost výsledků MeSH
- RNA rostlin izolace a purifikace MeSH
- rostlinné proteiny izolace a purifikace MeSH
- rostliny * genetika metabolismus MeSH
- systémová biologie metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Incubation and pot experiments using poplar (Populus nigra L. cv. Wolterson) were performed in order to evaluate the questionable efficiency of EDDS-enhanced phytoextraction of Cu from contaminated soils. Despite the promising conditions of the experiment (low contamination of soils with a single metal with a high affinity for EDDS, metal tolerant poplar species capable of producing high biomass yields, root colonization by mycorrhizal fungi), the phytoextraction efficiency was not sufficient. The EDDS concentrations used in this study (3 and 6 mmol kg(-1)) enhanced the mobility (up to a 100-fold increase) and plant uptake of Cu (up to a 65-fold increase). However, despite EDDS degradation and the competition of Fe and Al for the chelant, Cu leaching cannot be omitted during the process. Due to the low efficiency, further research should be focused on other environment-friendly methods of soil remediation.
Chelant-enhanced phytoextraction of heavy metals is an emerging technological approach for a non-destructive remediation of contaminated soils. The main objectives of this study were (i) to assess the extraction efficiency of two different synthetic chelating agents (ethylenediaminetetraacetic acid (EDTA) and ethylenediaminedisuccinic acid (EDDS)) for desorbing Pb from two contaminated agricultural soils originating from a mining and smelting district and (ii) to assess the phytoextraction efficiency of maize (Zea mays) and poplar (Populus sp.) after EDTA application. EDTA was more efficient than EDDS in desorbing and complexing Pb from both soils, removing as much as 60% of Pb. Maize exhibited better results than poplar when extracting Pb from the more acidic (pH approximately 4) and more contaminated (up to 1360 mg Pb kg(-1)) agricultural soil originating from the smelting area. On the other hand, poplars proved to be more efficient when grown on the near-neutral (pH approximately 6) and less contaminated (up to 200 mg Pb kg(-1)) agricultural soil originating from the mining area. Furthermore, the addition of EDTA led to a significant increase of Pb content especially in poplar leaves, proving a strong translocation rate within the poplar plants.
- MeSH
- biodegradace MeSH
- biomasa MeSH
- chelátory chemie MeSH
- EDTA chemie MeSH
- ethylendiaminy chemie MeSH
- financování organizované MeSH
- hornictví MeSH
- kukuřice setá chemie metabolismus MeSH
- látky znečišťující půdu izolace a purifikace MeSH
- olovo izolace a purifikace metabolismus MeSH
- Populus chemie metabolismus MeSH
- průmyslový odpad škodlivé účinky MeSH
- půda analýza MeSH
- sukcináty chemie MeSH
- zemědělství MeSH
