| 1. |
- Sandh, Gustaf, et al.
(författare)
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Comparative proteomic profiles of the marine cyanobacterium Trichodesmium erythraeum IMS101 under different nitrogen regimes
- 2011
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Ingår i: Proteomics. - : Wiley. - 1615-9853 .- 1615-9861. ; 11:3, s. 406-419
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Tidskriftsartikel (refereegranskat)abstract
- Trichodesmium is a marine filamentous diazotrophic cyanobacterium and an important contributor of "new" nitrogen in the oligotrophic surface waters of the tropical and subtropical oceans. It is unique in that it exclusively fixes N-2 at daytime, although it belongs to the non-heterocystous filamentous segment of the cyanobacterial radiation. Here we present the first quantitative proteomic analysis of Trichodesmium erythraeum IMS101 when grown under different nitrogen regimes using 2-DE/MALDI-TOF-MS. Addition of combined nitrogen (NO3-) prevented development of the morphological characteristics of the N-2-fixing cell type (diazocytes), inhibited expression of the nitrogenase enzyme subunits and consequently N-2 fixation activity. The diazotrophic regime (N-2 versus NO3- cultures) elicited the differential expression of more than 100 proteins, which represented 13.5% of the separated proteins. Besides proteins directly related to N-2 fixation, proteins involved in the synthesis of reducing equivalents and the generation of a micro-oxic environment were strongly up-regulated, as was in particular Dps, a protein related to iron acquisition and potentially other vital cellular processes. In contrast, proteins involved in the S-adenosylmethionine (SAM) cycle, synthesis of amino acids and production of carbon skeletons for storage and synthesis of amino acids were suppressed. The data are discussed in the context of Trichodesmium's unusual N-2-fixing physiology.
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| 2. |
- Cifuentes, Carolina, et al.
(författare)
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Biosynthesis of Callose and Cellulose by Detergent Extracts of Tobacco Cell Membranes and Quantification of the Polymers Synthesized in vitro
- 2010
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Ingår i: J INTEGR PLANT BIOL. - : Wiley. - 1672-9072. ; 52:2, s. 221-233
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Tidskriftsartikel (refereegranskat)abstract
- The conditions that favor the in vitro synthesis of cellulose from tobacco BY-2 cell extracts were determined. The procedure leading to the highest yield of cellulose consisted of incubating digitonin extracts of membranes from 11-day-old tobacco BY-2 cells in the presence of 1 mM UDP-glucose, 8 mM Ca2+ and 8 mM Mg2+. Under these conditions, up to nearly 40% of the polysaccharides synthesized in vitro corresponded to cellulose, the other polymer synthesized being callose. Transmission electron microscopy analysis revealed the occurrence of two types of structures in the synthetic reactions. The first type consisted of small aggregates with a diameter between 3 and 5 nm that associated to form fibrillar strings of a maximum length of 400 nm. These structures were sensitive to the acetic/nitric acid treatment of Updegraff and corresponded to callose. The second type of structures was resistant to the Updegraff reagent and corresponded to straight cellulose microfibrils of 2-3 nm in diameter and 200 nm to up to 5 mu m in length. In vitro reactions performed on electron microscopy grids indicated that the minimal rate of microfibril elongation in vitro is 120 nm/min. Measurements of retardance by liquid crystal polarization microscopy as a function of time showed that small groups of microfibrils increased in retardance by up to 0.047 nm/min per pixel, confirming the formation of organized structures.
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| 3. |
- Guerriero, Gea, et al.
(författare)
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What Do We Really Know about Cellulose Biosynthesis in Higher Plants?
- 2010
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Ingår i: JOURNAL OF INTEGRATIVE PLANT BIOLOGY. - : Wiley. - 1672-9072 .- 1744-7909. ; 52:2, s. 161-175
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Forskningsöversikt (refereegranskat)abstract
- Cellulose biosynthesis is one of the most important biochemical processes in plant biology. Despite the considerable progress made during the last decade, numerous fundamental questions related to this key process in plant development are outstanding. Numerous models have been proposed through the years to explain the detailed molecular events of cellulose biosynthesis. Almost all models integrate solid experimental data with hypotheses on several of the steps involved in the process. Speculative models are most useful to stimulate further research investigations and bring new exciting ideas to the field. However, it is important to keep their hypothetical nature in mind and be aware of the risk that some undemonstrated hypotheses may progressively become admitted. In this review, we discuss the different steps required for cellulose formation and crystallization, and highlight the most important specific aspects that are supported by solid experimental data.
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| 4. |
- Larsbrink, Johan, 1982, et al.
(författare)
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Proteomic data on enzyme secretion and activity in the bacterium Chitinophaga pinensis
- 2017
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Ingår i: Data in Brief. - : Elsevier BV. - 2352-3409. ; 11, s. 484-490
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Tidskriftsartikel (refereegranskat)abstract
- The secretion of carbohydrate-degrading enzymes by a bacterium sourced from a softwood forest environment has been investigated by mass spectrometry. The findings are discussed in full in the research article “Proteomic insights into mannan degradation and protein secretion by the forest floor bacterium Chitinophaga pinensis” in Journal of Proteomics by Larsbrink et al. ([1], doi: 10.1016/j.jprot.2017.01.003). The bacterium was grown on three carbon sources (glucose, glucomannan, and galactomannan) which are likely to be nutrient sources or carbohydrate degradation products found in its natural habitat. The bacterium was grown on solid agarose plates to mimic the natural behaviour of growth on a solid surface. Secreted proteins were collected from the agarose following trypsin-mediated hydrolysis to peptides. The different carbon sources led to the secretion of different numbers and types of proteins. Most carbohydrate-degrading enzymes were found in the glucomannan-induced cultures. Several of these enzymes may have biotechnological potential in plant cell wall deconstruction for biofuel or biomaterial production, and several may have novel activities. A subset of carbohydrate-active enzymes (CAZymes) with predicted activities not obviously related to the growth substrates were also found in samples grown on each of the three carbohydrates. The full dataset is accessible at the PRIDE partner repository (ProteomeXchange Consortium) with the identifier PXD004305, and the full list of proteins detected is given in the supplementary material attached to this report.
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| 5. |
- Klinter, Stefan, 1985-
(författare)
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Identification and characterisation of chitin and cellulose synthases in oomycetes : New tools for biochemical studies and structure determination
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Despite resembling ‘true’ fungi in terms of morphological features, oomycetes form a distinct eukaryotic lineage of filamentous microorganisms that belongs to the stramenopiles, a group of protists also comprising the closely-related brown algae and diatoms. Many oomycetes are devastating pathogens of plants and animals, globally causing significant economic losses in the agriculture and aquaculture industries, and posing considerable environmental damage to natural ecosystems. Although the cell wall (CW) is critical for the viability and morphogenesis of the organism it surrounds, our knowledge of oomycete CW architecture and biosynthetic enzymes is limited. Given the vast threat that pathogenic oomycetes pose, uncovering the details of CW biosynthesis and regulation in these pathogens may reveal new opportunities for disease control.To this end, we aimed to elucidate the role of putative membrane-bound glycosyltransferase family 2 enzymes implicated in the biosynthesis of oomycete CW polysaccharides. Suitable gene candidates were identified, and their products analysed, as illustrated by the oomycete-wide discovery and phylogenetic analysis of the chitin synthase gene family (paper I), and the identification of the cellulose synthase genes in Saprolegnia parasitica (paper II) and Phytophthora capsici (paper III). Expression of promising candidate genes was verified using different techniques, including gene expression analysis (papers II and III), and the effect of inhibitors on hyphal growth (papers I and II) and enzymatic activity in in vitro assays (paper II). Single enzymes representing putative chitin synthases from various organisms (unpublished data) and cellulose synthases from S. parasitica (extended data for paper II), and P. capsici cellulose synthase 1 (paper III) were produced, and partly enriched or even purified, in yeast strains specifically engineered to facilitate the biochemical characterisation of the recombinant proteins in in vitro enzyme assays. To advance functional investigations and structure determination of integral membrane proteins, we developed DirectMX, a method that allows the reconstitution of target proteins with their surrounding lipids directly from crude cell membranes into Salipro nanoparticles (paper IV).
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| 6. |
- Jawaid, Muhammad Zaki, et al.
(författare)
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A biophysical model for plant cell plate maturation based on the contribution of a spreading force
- 2022
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Ingår i: Plant Physiology. - : Oxford University Press (OUP). - 0032-0889 .- 1532-2548. ; 188:2, s. 795-806
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Tidskriftsartikel (refereegranskat)abstract
- The late-stage onset of an "areal" spreading and stabilizing force is essential for regular plant cell plate development and maturation. Plant cytokinesis, a fundamental process of plant life, involves de novo formation of a "cell plate" partitioning the cytoplasm of dividing cells. Cell plate formation is directed by orchestrated delivery, fusion of cytokinetic vesicles, and membrane maturation to form a nascent cell wall by timely deposition of polysaccharides. During cell plate maturation, the fragile membrane network transitions to a fenestrated sheet and finally a young cell wall. Here, we approximated cell plate sub-structures with testable shapes and adopted the Helfrich-free energy model for membranes, including a stabilizing and spreading force, to understand the transition from a vesicular network to a fenestrated sheet and mature cell plate. Regular cell plate development in the model was possible, with suitable bending modulus, for a two-dimensional late stage spreading force of 2-6 pN/nm, an osmotic pressure difference of 2-10 kPa, and spontaneous curvature between 0 and 0.04 nm(-1). With these conditions, stable membrane conformation sizes and morphologies emerged in concordance with stages of cell plate development. To reach a mature cell plate, our model required the late-stage onset of a spreading/stabilizing force coupled with a concurrent loss of spontaneous curvature. Absence of a spreading/stabilizing force predicts failure of maturation. The proposed model provides a framework to interrogate different players in late cytokinesis and potentially other membrane networks that undergo such transitions. Callose, is a polysaccharide that accumulates transiently during cell plate maturation. Callose-related observations were consistent with the proposed model's concept, suggesting that it is one of the factors involved in establishing the spreading force.
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| 7. |
- Malm, Erik, et al.
(författare)
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The surface structure of well-ordered native cellulose fibrils in contact with water
- 2010
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Ingår i: Carbohydrate Research. - : Elsevier BV. - 0008-6215 .- 1873-426X. ; 345:1, s. 97-100
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Tidskriftsartikel (refereegranskat)abstract
- CP/MAS C-13 NMR spectroscopy was used in combination with spectral fitting to examine the surface structure of hydrated cellulose I fibrils from Halocynthia and Gluconoacetobacter xylinus. To increase the spectral intensities and minimize signal overlap, G. xylinus celluloses site-specifically enriched in C-13 either on C4 or on both C1 and C6 were examined. The experimental data showed multiple C4 and C6 signals for the water accessible fibril surfaces in the highly crystalline celluloses. These signal multiplicities were attributed to structural features in the surface layers induced by the fibril interior, and could not be extracted by spectral fitting in celluloses with a lower degree of crystallinity such as cellulose from cotton.
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| 8. |
- Lou, H., et al.
(författare)
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The cellulose synthase-like F3 (CslF3) gene mediates cell wall polysaccharide synthesis and affects root growth and differentiation in barley
- 2022
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Ingår i: The Plant Journal. - : Wiley. - 0960-7412 .- 1365-313X. ; 110:6, s. 1681-1699
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Tidskriftsartikel (refereegranskat)abstract
- The barley cellulose synthase-like F (CslF) genes encode putative cell wall polysaccharide synthases. They are related to the cellulose synthase (CesA) genes involved in cellulose biosynthesis, and the CslD genes that influence root hair development. Although CslD genes are implicated in callose, mannan and cellulose biosynthesis, and are found in both monocots and eudicots, CslF genes are specific to the Poaceae. Recently the barley CslF3 (HvCslF3) gene was shown to be involved in the synthesis of a novel (1,4)-β-linked glucoxylan, but it remains unclear whether this gene contributes to plant growth and development. Here, expression profiling using qRT-PCR and mRNA in situ hybridization revealed that HvCslF3 accumulates in the root elongation zone. Silencing HvCslF3 by RNAi was accompanied by slower root growth, linked with a shorter elongation zone and a significant reduction in root system size. Polymer profiling of the RNAi lines revealed a significant reduction in (1,4)-β-linked glucoxylan levels. Remarkably, the heterologous expression of HvCslF3 in wild-type (Col-0) and root hair-deficient Arabidopsis mutants (csld3 and csld5) complemented the csld5 mutant phenotype, in addition to altering epidermal cell fate. Our results reveal a key role for HvCslF3 during barley root development and suggest that members of the CslD and CslF gene families have similar functions during root growth regulation.
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| 9. |
- Roberts, Alison W., et al.
(författare)
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Functional Characterization of a Glycosyltransferase from the Moss Physcomitrella patens Involved in the Biosynthesis of a Novel Cell Wall Arabinoglucan
- 2018
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Ingår i: The Plant Cell. - : American Society of Plant Biologists. - 1040-4651 .- 1532-298X. ; 30:6, s. 1293-1308
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Tidskriftsartikel (refereegranskat)abstract
- Mixed-linkage (1,3;1,4)-β-glucan (MLG), an abundant cell wall polysaccharide in the Poaceae, has been detected in ascomycetes, algae, and seedless vascular plants, but not in eudicots. Although MLG has not been reported in bryophytes, a predicted glycosyltransferase from the moss Physcomitrella patens (Pp3c12_24670) is similar to a bona fide ascomycete MLG synthase. We tested whether Pp3c12_24670 encodes an MLG synthase by expressing it in wild tobacco (Nicotiana benthamiana) and testing for release of diagnostic oligosaccharides from the cell walls by either lichenase or (1,4)-β-glucan endohydrolase. Lichenase, an MLG-specific endohydrolase, showed no activity against cell walls from transformed N. benthamiana, but (1,4)-β-glucan endohydrolase released oligosaccharides that were distinct from oligosaccharides released from MLG by this enzyme. Further analysis revealed that these oligosaccharides were derived from a novel unbranched, unsubstituted arabinoglucan (AGlc) polysaccharide. We identified sequences similar to the P. patens AGlc synthase from algae, bryophytes, lycophytes, and monilophytes, raising the possibility that other early divergent plants synthesize AGlc. Similarity of P. patens AGlc synthase to MLG synthases from ascomycetes, but not those from Poaceae, suggests that AGlc and MLG have a common evolutionary history that includes loss in seed plants, followed by a more recent independent origin of MLG within the monocots.
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| 10. |
- Kumar, Rajender, et al.
(författare)
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Biochemical characterization and molecular insights into substrate recognition of pectin methylesterase from Phytophthora infestans
- 2022
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Ingår i: Computational and Structural Biotechnology Journal. - : Elsevier BV. - 2001-0370. ; 20, s. 6023-6032
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Tidskriftsartikel (refereegranskat)abstract
- Pectin methylesterases (PMEs) are a class of carbohydrate-active enzymes that act on the O6-methyl ester groups of the homogalacturonan component of pectins, resulting in de-esterification of the sub-strate polymers and formation of pectate and methanol. PMEs occur in higher plants and microorgan-isms, including fungi, oomycetes, bacteria, and archaea. Microbial PMEs play a crucial role in pathogens' invasion of plant tissues. Here, we have determined the structural and functional properties of Pi-PME, a PME from the oomycete plant pathogen Phytophthora infestans. This enzyme exhibits maxi-mum activity at alkaline pH (8.5) and is active over a wide temperature range (25-50 degrees C). In silico deter-mination of the structure of Pi-PME reveals that the protein consists essentially of three parallel 8-sheets interconnected by loops that adopt an overall 8-helix organization. The loop regions in the vicinity of the active site are extended compared to plant and fungal PMEs, but they are shorter than the corresponding bacterial and insect regions. Molecular dynamic simulations revealed that Pi-PME interacts most strongly with partially de-methylated homogalacturonans, suggesting that it preferentially uses this type of sub-strates. The results are compared and discussed with other known PMEs from different organisms, high-lighting the specific features of Pi-PME.
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