| 1. |
- Blaschek, Leonard
(author)
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Cellular Lignin Distribution Patterns and their Physiological Relevance
- 2020
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Licentiate thesis (other academic/artistic)abstract
- The diverse morphological shapes of plants are made possible by the structural rigidity provided by cell walls. In order to support vertical growth and long distance water transport, cell walls need to resist a variety of biological and physical stresses. Lignin, a cell wall polyphenolic unique to vascular plants, has long been considered to structurally support the cell walls of xylem vessels and other specialised cell types against these forces. Lignin is a complex polymer whose monomeric composition and biochemical properties vary widely between different species, tissues and cell types. However, the precise characterisation of this micro-scale variation poses considerable methodological hurdles. As a result, it has yet to be understood how differences in lignin composition contribute to the cell-type specific functions of the cell wall. In the works presented herein, we optimise and validate the Wiesner test and Raman microspectroscopy for the quantitative characterisation of lignin in situ and use these techniques to show how cell-type specific genetic regulation of lignification is crucial for cell wall function. Using synthetic lignin monomers and polymers, as well as genetically altered Arabidopsis and Populus plants in conjunction with biochemical lignin composition analyses, we establish the Wiesner test as a specific high-resolution method to quantify coniferaldehyde (I), and show that Raman microspectroscopy allows the relative quantification of total lignin, guaiacyl lignin subunits (G-units), coniferyl alcohol and syringyl lignin subunits (S-units) (II). We then use these methods to characterise cell-autonomous and cell-cell cooperative lignification patterns and show that cell walls of different vessel types depend on distinct amounts of lignin and specific G-units for structural reinforcement (III). S-unit incorporation into vessel lignin and increased adjacency to neighbouring vessels on the other hand compromise their resistance to collapse (III). Altogether, we provide evidence for a lignification process consisting of a fine scale, cell-type specific regulatory network of lignin biosynthesis, cell-to-cell cooperative monomer supply, and cell wall layer specific monomer incorporation. Crucially, it is this dynamic small-scale regulation that allows lignified plant cell walls to fulfil their cell-type specific functions.
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| 2. |
- Chambi, Diego, et al.
(author)
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Production of Exopolysaccharides by Cultivation of Halotolerant Bacillus atrophaeus BU4 in Glucose- and Xylose-Based Synthetic Media and in Hydrolysates of Quinoa Stalks
- 2022
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In: Fermentation. - : MDPI AG. - 2311-5637. ; 8:2
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Journal article (peer-reviewed)abstract
- A halotolerant, exopolysaccharide-producing bacterium isolated from the Salar de Uyuni salt flat in Bolivia was identified as Bacillus atrophaeus using next-generation sequencing. Comparisons indicate that the genome most likely (p-value: 0.0024) belongs to a subspecies previously not represented in the database. The growth of the bacterial strain and its ability to produce exopolysaccharides (EPS) in synthetic media with glucose or xylose as carbon sources, and in hydrolysates of quinoa stalks, was investigated. The strain grew well in all synthetic media, but the growth in glucose was better than that in xylose. Sugar consumption was better when initial concentrations were low. The growth was good in enzymatically produced cellulosic hydrolysates but was inhibited in hemicellulosic hydrolysates produced using hydrothermal pretreatment. The EPS yields were up to 0.064 g/g on initial glucose and 0.047 g/g on initial xylose, and was higher in media with relatively low sugar concentrations. The EPS was isolated and purified by a sequential procedure including centrifugation, cold ethanol precipitation, trichloroacetic acid treatment, dialysis, and freeze-drying. Glucose and mannose were the main sugars identified in hydrolyzed EPS. The EPS was characterized by size-exclusion chromatography, Fourier-transform infrared (FTIR) spectroscopy, heteronuclear single-quantum coherence nuclear magnetic resonance (HSQC NMR) spectroscopy, scanning electron microscopy, X-ray diffraction, and thermogravimetric analysis. No major differences were elucidated between EPS resulting from cultivations in glucose- or-xylose-based synthetic media, while some divergences with regard to molecular-weight averages and FTIR and HSQC NMR spectra were detected for EPS from hydrolysate-based media.
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| 3. |
- Funda, Tomas, et al.
(author)
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Predicting the chemical composition of juvenile and mature woods in Scots pine (Pinus sylvestris L.) using FTIR spectroscopy
- 2020
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In: Wood Science and Technology. - : Springer. - 0043-7719 .- 1432-5225. ; 54, s. 289-311
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Journal article (peer-reviewed)abstract
- The chemical composition of wood is one of the key features that determine wood quality. The focus of this study was on identifying differences between juvenile and mature woods in Scots pine (Pinus sylvestris L.) and developing models for predicting the chemical composition of these two wood types. Chemical traits, determined by traditional wet chemistry techniques, included the proportion of lignin, polysaccharides and extractives. Partial least squares regression of Fourier transform infrared (FTIR) spectra was used for model building. The model performance was primarily evaluated by root mean squared error of predictions (RMSEP). High predictive power was attained for the content of lignin (RMSEP of 0.476 and 0.495 for juvenile and mature woods, respectively) and extractives (0.302 and 0.471), good predictive power for cellulose (0.715 and 0.696) and hemicelluloses in juvenile wood (0.719) and low predictive power for hemicelluloses in mature wood (0.823). A distinct band was observed at 1693 cm(-1), and its intensity was strongly associated with the content of extractives (r = 0.968 and 0.861 in juvenile and mature woods, respectively). FTIR has proved suitable for the rapid, non-destructive, cost-efficient assessment of the chemical composition of juvenile and mature woods in Scots pine. The band at 1693 cm(-1) is to be further investigated to unravel its link with individual extractive components.
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| 4. |
- Guillon, Fabienne, et al.
(author)
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In situ imaging of lignin and related compounds by Raman, Fourier-transform infrared (FTIR) and fluorescence microscopy
- 2022
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In: Advances in Botanical Research. - : Elsevier. - 0065-2296 .- 2162-5948. ; 104, s. 215-270
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Journal article (peer-reviewed)abstract
- Spectral imaging technologies simultaneously record spectral and spatial information about plant tissues in a noninvasive way. Differences in techniques result in different selection rules and spatial resolutions. This article introduces the basic principles of Raman, Fourier-transform Infrared (FTIR) and autofluorescence imaging and finally compares their strength and drawbacks. The methods result in spectral datasets as bases for image generation. Spectral preprocessing together with univariate and multivariate data analysis approaches are essential for informative lignin imaging and analysis and therefore also briefly illustrated and discussed. Examples of imaging lignin and other aromatic components in a broad range of plant tissues show the potential as well as the limitations of microspectroscopic imaging.
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| 5. |
- Hahn, Max, 1993-
(author)
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Characterizing the pancreatic "isletome" : 3D optical imaging to study diabetes
- 2022
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Doctoral thesis (other academic/artistic)abstract
- The pancreas is a specialised multipurpose organ, that can be separated into two major compartments: endocrine and exocrine. The exocrine part makes up the majority of the organ volume and functions to secrete digestive enzymes into the small intestine. Notably, endocrine islets of Langerhans are embedded and scattered in vast numbers throughout the exocrine space. These miniature functional units are composed of different cell types that secrete hormones into the blood stream. The most abundant islet-cell is the insulin-producing β-cell. Highly coordinated, the endocrine cells are the primary regulators of energy homeostasis in the body. Together, the collective islet volume constitutes the pancreatic “isletome”, a synchronised, complex and size-equilibrated system that is able to respond to various metabolic conditions. Indeed, environmental and/or genetic conditions often lead to impaired islet function and/or β-cell destruction leading to elevated blood glucose levels over time and eventually diabetes. Diabetes mellitus is a disease that currently affects more than 400 million individuals worldwide. As such, understanding pancreatic disease-related mechanisms is pivotal to the development of new and more effective therapeutic, or even curative, regimens. The deep location of the pancreas in the abdomen and the relatively low resolution of current clinical imaging approaches, however, render the pancreatic islets difficult to study when visually assessing endocrine function. Although non-invasive imaging techniques have yet to reach their full potential, post-mortem studies of the pancreas and rodent disease models offer unique insights into the process of diabetes disease dynamics.Diabetes induced by streptozotocin (STZ) is a widely used model system in pre-clinical research, where it is generally believed that the b-cells are depleted upon the administration of the drug. Yet, quantification of β-cell volume dynamics and underlying disease mechanisms have not been extensively described. Using optical projection tomography (OPT), light sheet fluorescence microscopy (LSFM) and advanced protocols for ex vivo whole organ three-dimensional (3D) imaging, this study demonstrated that STZ-induced β-cell depletion is modest, primarily affecting large islets, and is not the primary cause for the development of diabetes in STZ-diabetic mice. Combined with islet gene expression studies, the remaining β-cell volume in STZ-diabetic mice displayed a downregulation of glucose transporter type 2 (GLUT2), a transmembrane carrier vital for sensing blood glucose levels. Islet transplantation into the anterior chamber of the eye (ACE) reversed the STZ-induced hyperglycaemia and partially restored islet function, including GLUT2, but did not restore β-cell volume loss. Extensive 3D image datasets were generated as a resource to the research community. The combined results of this study indicated that STZ-induced hyperglycaemia is not caused by β-cell loss, but rather by dysfunctional β-cells and that recovery of islet function is restrained by continuous hyperglycaemia.3D imaging using OPT has proven to be a reliable technique in quantifying cellular/anatomical features of the mouse pancreas. However, the technique has rarely been applied to patient-derived tissues. Here, a label-free and non-destructive method was developed to assess clinical biopsies within hours of collection. Specifically, this study showed that autofluorescence-based imaging can be used to delineate tumours of the pancreas (pancreatic ductal adenocarcinoma, PDAC) in 3D, which may aid in identifying tumour margins in conjunction with resective surgery. Importantly, the protocol included a reversal pipeline so that other histological workflows could be applied to the same specimen. Furthermore, this study demonstrated that natural fluorescent substances in the endocrine cells provide sufficient contrast when quantifying both the volume and number of islets of Langerhans in the healthy pancreas. Altogether, the developed technique may provide a novel tool for the rapid 3D analysis of pancreatic biopsies that may complement and improve traditional pathological assessments.With the emergence of islet transplantation networks worldwide, access to fixed pancreatic tissues from diseased donors has dramatically improved. Hereby, the near instant autolysis of the pancreas post-mortem can generally be avoided, which provides the opportunity to quantitatively study the entire gland ex vivo within a conserved spatial context. Yet, mesoscopic 3D imaging of the pancreas (by OPT and/or LSFM) has been limited predominantly due to the obstacle of labelling larger tissue volumes. As such, a simple approach to antibody labelling and cellular imaging was developed in cubic centimetre-sized tissue cuboids that were mapped to the whole organ. By stitching the resultant datasets back into 3D space, this approach demonstrated how essentially any human organ may be analysed in full with high resolution. This technique was applied to pancreata from non-diabetic and type 2 diabetic (T2D) donors, analysing over 200 thousand islets, revealing features of the human pancreas that were not analysed in 3D previously, including high islet dense regions and intra-islet haemorrhaging. Crucially, this new technique may contribute to unveil a wealth of new insights into the complex pathophysiology of the “diabetic pancreas”.By applying the above method to the entire volume of the human pancreas, the absolute distribution and volume of insulin-positive cells in a pancreas from a donor with longstanding type 1 diabetes (T1D) was demonstrated for the first time. By dividing the 19 cm long organ into smaller pieces, followed by insulin labelling, OPT imaging and reconstruction in 3D space, approximately 173,000 insulin-positive objects were identified. By utilising tissue autofluorescence, the entire organ was reconstructed in 3D, together with blood vessels and ducts. These data indicated several important regional differences in β-cell mass, such as the uncinate process showing the highest density, which potentially reflects key aspects of disease dynamics. Furthermore, regions with a “punctated distribution” of single β-cells in close proximity to each other were identified. Although the significance of these observations needs to be elucidated, we speculate that these regions could be associated with pancreatic regeneration, which might permit the development of new interventions for clinical regenerative processes in the future. Altogether, this study represents the first whole organ account of β-cell distribution at the current level of resolution in an entire organ. As such, it may serve as an important advancement towards detailed whole organ analyses of endocrine cell identity/function, via a wide range of markers, in the study of normal anatomy and pathophysiology of the human pancreas.
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| 6. |
- Hurný, Andrej, et al.
(author)
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SYNERGISTIC ON AUXIN AND CYTOKININ 1 positively regulates growth and attenuates soil pathogen resistance
- 2020
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In: Nature Communications. - : Nature Publishing Group. - 2041-1723. ; 11:1, s. 1-17
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Journal article (peer-reviewed)abstract
- Plants as non-mobile organisms constantly integrate varying environmental signals to flexibly adapt their growth and development. Local fluctuations in water and nutrient availability, sudden changes in temperature or other abiotic and biotic stresses can trigger changes in the growth of plant organs. Multiple mutually interconnected hormonal signaling cascades act as essential endogenous translators of these exogenous signals in the adaptive responses of plants. Although the molecular backbones of hormone transduction pathways have been identified, the mechanisms underlying their interactions are largely unknown. Here, using genome wide transcriptome profiling we identify an auxin and cytokinin cross-talk component; SYNERGISTIC ON AUXIN AND CYTOKININ 1 (SYAC1), whose expression in roots is strictly dependent on both of these hormonal pathways. We show that SYAC1 is a regulator of secretory pathway, whose enhanced activity interferes with deposition of cell wall components and can fine-tune organ growth and sensitivity to soil pathogens. Cytokinin and auxin are two major hormonal regulators of plant growth. Here the authors identify SYAC1, a gene that is synergistically activated by the two hormones being applied together, and show that it is required for normal growth while negatively impacting pathogen resistance.
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| 7. |
- Kushwah, Sunita, et al.
(author)
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Arabidopsis XTH4 and XTH9 Contribute to Wood Cell Expansion and Secondary Wall Formation(1)([OPEN])
- 2020
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In: Plant Physiology. - : American Society of Plant Science. - 0032-0889 .- 1532-2548. ; 182:4, s. 1946-1965
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Journal article (peer-reviewed)abstract
- Xyloglucan is the major hemicellulose of dicotyledon primary cell walls, affecting the load-bearing framework with the participation of xyloglucan endo-transglycosylase/hydrolases (XTHs). We used loss- and gain-of function approaches to study functions of XTH4 and XTH9 abundantly expressed in cambial regions during secondary growth of Arabidopsis (Arabidopsis thaliana). In secondarily thickened hypocotyls, these enzymes had positive effects on vessel element expansion and fiber intrusive growth. They also stimulated secondary wall thickening but reduced secondary xylem production. Cell wall analyses of inflorescence stems revealed changes in lignin, cellulose, and matrix sugar composition indicating an overall increase in secondary versus primary walls in mutants, indicative of higher xylem production compared with the wild type (since secondary walls were thinner). Intriguingly, the number of secondary cell wall layers compared with the wild type was increased in xth9 and reduced in xth4, whereas the double mutant xth4x9 displayed an intermediate number of layers. These changes correlated with specific Raman signals from the walls, indicating changes in lignin and cellulose. Secondary walls were affected also in the interfascicular fibers, where neither XTH4 nor XTH9 was expressed, indicating that these effects were indirect. Transcripts involved in secondary wall biosynthesis and cell wall integrity sensing, including THESEUS1 and WALL ASSOCIATED KINASE2, were highly induced in the mutants, indicating that deficiency in XTH4 and XTH9 triggers cell wall integrity signaling, which, we propose, stimulates xylem cell production and modulates secondary wall thickening. Prominent effects of XTH4 and XTH9 on secondary xylem support the hypothesis that altered xyloglucan affects wood properties both directly and via cell wall integrity sensing.
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| 8. |
- Marinovíc, Mila, et al.
(author)
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Comparative Analysis of Enzyme Production Patterns of Lignocellulose Degradation of Two White Rot Fungi : Obba rivulosa and Gelatoporia subvermispora
- 2022
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In: Biomolecules. - : MDPI. - 2218-273X. ; 12:8
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Journal article (peer-reviewed)abstract
- The unique ability of basidiomycete white rot fungi to degrade all components of plant cell walls makes them indispensable organisms in the global carbon cycle. In this study, we analyzed the proteomes of two closely related white rot fungi, Obba rivulosa and Gelatoporia subvermispora, during eight-week cultivation on solid spruce wood. Plant cell wall degrading carbohydrate-active enzymes (CAZymes) represented approximately 5% of the total proteins in both species. A core set of orthologous plant cell wall degrading CAZymes was shared between these species on spruce suggesting a conserved plant biomass degradation approach in this clade of basidiomycete fungi. However, differences in time-dependent production of plant cell wall degrading enzymes may be due to differences among initial growth rates of these species on solid spruce wood. The obtained results provide insight into specific enzymes and enzyme sets that are produced during the degradation of solid spruce wood in these fungi. These findings expand the knowledge on enzyme production in nature-mimicking conditions and may contribute to the exploitation of white rot fungi and their enzymes for biotechnological applications.
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| 9. |
- Miranda, Diego A., et al.
(author)
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Production and characterization of poly(3-hydroxybutyrate) from Halomonas boliviensis LC1 cultivated in hydrolysates of quinoa stalks
- 2023
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In: Fermentation. - : MDPI. - 2311-5637. ; 9:6
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Journal article (peer-reviewed)abstract
- The global production of fossil-based plastics has reached critical levels, and their substitution with bio-based polymers is an urgent requirement. Poly(3-hydroxybutyrate) (PHB) is a biopolymer that can be produced via microbial cultivation, but efficient microorganisms and low-cost substrates are required. Halomonas boliviensis LC1, a moderately halophilic bacterium, is an effective PHB producer, and hydrolysates of the residual stalks of quinoa (Chenopodium quinoa Willd.) can be considered a cheap source of sugars for microbial fermentation processes in quinoa-producing countries. In this study, H. boliviensis LC1 was adapted to a cellulosic hydrolysate of quinoa stalks obtained via acid-catalyzed hydrothermal pretreatment and enzymatic saccharification. The adapted strain was cultivated in hydrolysates and synthetic media, each of them with two different initial concentrations of glucose. Cell growth, glucose consumption, and PHB formation during cultivation were assessed. The cultivation results showed an initial lag in microbial growth and glucose consumption in the quinoa hydrolysates compared to cultivation in synthetic medium, but after 33 h, the values were comparable for all media. Cultivation in hydrolysates with an initial glucose concentration of 15 g/L resulted in a higher glucose consumption rate (0.15 g/(L h) vs. 0.14 g/(L h)) and volumetric productivity of PHB (14.02 mg/(L h) vs. 10.89 mg/(L h)) than cultivation in hydrolysates with 20 g/L as the initial glucose concentration. During most of the cultivation time, the PHB yield on initial glucose was higher for cultivation in synthetic medium than in hydrolysates. The produced PHBs were characterized using advanced analytical techniques, such as high-performance size-exclusion chromatography (HPSEC), Fourier transform infrared (FTIR) spectroscopy, 1H nuclear magnetic resonance (NMR) spectroscopy, scanning electron microscopy (SEM), X-ray diffraction (XRD), and thermogravimetric analysis (TGA). HPSEC revealed that the molecular weight of PHB produced in the cellulosic hydrolysate was lower than that of PHB produced in synthetic medium. TGA showed higher thermal stability for PHB produced in synthetic medium than for that produced in the hydrolysate. The results of the other characterization techniques displayed comparable features for both PHB samples. The presented results show the feasibility of producing PHB from quinoa stalks with H. boliviensis.
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| 10. |
- Niemi, Calle, et al.
(author)
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Quantitative and qualitative saccharide analysis of North Atlantic brown seaweed by gas chromatography/mass spectrometry and infrared spectroscopy
- 2024
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In: International Journal of Biological Macromolecules. - : Elsevier. - 0141-8130 .- 1879-0003. ; 254
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Journal article (peer-reviewed)abstract
- Brown seaweeds contain a variety of saccharides which have potential industrial uses. The most abundant polysaccharide in brown seaweed is typically alginate, consisting of mannuronic (M) and guluronic acid (G). The ratio of these residues fundamentally determines the physicochemical properties of alginate. In the present study, gas chromatography/mass spectrometry (GC/MS) was used to give a detailed breakdown of the monosaccharide species in North Atlantic brown seaweeds. The anthrone method was used for determination of crystalline cellulose. The experimental data was used to calibrate multivariate prediction models for estimation of total carbohydrates, crystalline cellulose, total alginate and alginate M/G ratio directly in dried, brown seaweed using three types of infrared spectroscopy, using relative error (RE) as a measure of predictive accuracy. Diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) performed well for the estimation of total alginate (RE = 0.12, R2 = 0.82), and attenuated total reflectance (ATR) showed good prediction of M/G ratio (RE = 0.14, R2 = 0.86). Both DRIFTS, ATR and near infrared (NIR) were unable to predict crystalline cellulose and only DRIFTS performed better in determining total carbohydrates. Multivariate spectral analysis is a promising method for easy and rapid characterization of alginate and M/G ratio in seaweed.
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