Publications by authors named "Bodil Jørgensen"

43 Publications

A diverse member of the fungal Avr4 effector family interacts with de-esterified pectin in plant cell walls to disrupt their integrity.

Sci Adv 2021 May 7;7(19). Epub 2021 May 7.

Department of Plant Pathology, University of California, Davis, Davis, CA, USA.

Effectors are small, secreted proteins that promote pathogen virulence. Although key to microbial infections, unlocking the intrinsic function of effectors remains a challenge. We have previously shown that members of the fungal Avr4 effector family use a carbohydrate-binding module of family 14 (CBM14) to bind chitin in fungal cell walls and protect them from host chitinases during infection. Here, we show that gene duplication in the Avr4 family produced an Avr4-2 paralog with a previously unknown effector function. Specifically, we functionally characterize Avr4-2, a paralog of Avr4 in the tomato pathogen , and show that although it contains a CBM14 domain, it does not bind chitin or protect fungi against chitinases. Instead, Avr4-2 interacts with highly de-esterified pectin in the plant's middle lamellae or primary cell walls and interferes with Ca-mediated cross-linking at cell-cell junction zones, thus loosening the plant cell wall structure and synergizing the activity of pathogen secreted endo-polygalacturonases.
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http://dx.doi.org/10.1126/sciadv.abe0809DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8104879PMC
May 2021

Comprehensive Leaf Cell Wall Analysis Using Carbohydrate Microarrays Reveals Polysaccharide-Level Variation between Species with Differing Resistance to Downy Mildew.

Polymers (Basel) 2021 Apr 23;13(9). Epub 2021 Apr 23.

Center for Viticulture and Enology, Department of Plant Science, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China.

The cell wall acts as one of the first barriers of the plant against various biotic stressors. Previous studies have shown that alterations in wall polysaccharides may influence crop disease resistance. In the grapevine family, several native species (e.g., Chinese wild grapevine) show a naturally higher resistance to microbial pathogens than cultivated species (e.g., ), and this trait could be inherited through breeding. Despite the importance of the cell wall in plant immunity, there are currently no comprehensive cell wall profiles of grapevine leaves displaying differing resistance phenotypes, due to the complex nature of the cell wall and the limitations of analytical techniques available. In this study, the cutting-edge comprehensive carbohydrate microarray technology was applied to profile uninfected leaves of the susceptible cultivar ( cv. "Cabernet Sauvignon"), a resistant cultivar ( cv. "Shuanghong") and a hybrid offspring cross displaying moderate resistance. The microarray approach uses monoclonal antibodies, which recognize polysaccharides epitopes, and found that epitope abundances of highly esterified homogalacturonan (HG), xyloglucan (with XXXG motif), (galacto)(gluco)mannan and arabinogalactan protein (AGP) appeared to be positively correlated with the high resistance of cv. "Shuanghong" to mildew. The quantification work by gas chromatography did not reveal any significant differences for the monosaccharide constituents, suggesting that polysaccharide structural alterations may contribute more crucially to the resistance observed; this is again supported by the contact infrared spectroscopy of cell wall residues, revealing chemical functional group changes (e.g., esterification of pectin). The identification of certain wall polysaccharides that showed alterations could be further correlated with resistance to mildew. Data from the use of the hybrid material in this study have preliminarily suggested that these traits could be inherited and may be applied as potential structural biomarkers in future breeding work.
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http://dx.doi.org/10.3390/polym13091379DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8122933PMC
April 2021

A group-based real-time videoconferencing telerehabilitation programme in recently discharged geriatric patients: a feasibility study.

Eur Geriatr Med 2021 Feb 5. Epub 2021 Feb 5.

Department of Geriatrics, Aarhus University Hospital, Palle Juul-Jensens, Boulevard 99, 8200, Aarhus N, Denmark.

Purpose: Exercise at home and improvement in the ability to undertake daily tasks are highly valued by older people after hospitalisation. New telerehabilitation (TR) technologies make it possible to supervise and communicate with exercising participants through videoconferencing equipment. This technology has been shown to be both feasible and effective in Danish chronic obstructive pulmonary disease patients in terms of basic mobility, safety, social interactions and patient perception. This study sought to examine whether it was feasible to carry out TR through home exercises in groups.

Methods: Both medical and hip-fracture home-dwelling patients aged 65 years and older admitted to the Emergency Department (ED) and Department of Geriatrics for acute reasons were asked to participate in the study just before their discharge. The inclusion criteria were normal cognitive function, being dependent on a walking aid and computer users before hospitalisation.

Results: At discharge, 333 patients were consecutively screened for participation. Of those, 300 patients were excluded. Thirty-three patients met the inclusion criteria. They had a mean age of 82.3 years (± 7.8) and 76% were women. Nine patients agreed to participate, but seven withdrew. The most frequent explanation was exhaustion in the continuation of hospitalisation.

Conclusion: It was not possible to conduct a videoconference TR study in a geriatric population, as many were excluded and those who were eligible withdrew after inclusion. During the COVID-19 pandemic, TR may be an important tool for isolated older persons to hinder functional decline. Aspects such as recruitment procedures and IT solutions designed for older people must be considered.
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http://dx.doi.org/10.1007/s41999-020-00444-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7863611PMC
February 2021

Amylose/cellulose nanofiber composites for all-natural, fully biodegradable and flexible bioplastics.

Carbohydr Polym 2021 Feb 22;253:117277. Epub 2020 Oct 22.

Department of Plant and Environmental Sciences, University of Copenhagen, 1871, Frederiksberg C, Denmark. Electronic address:

Thermoplastic, polysaccharide-based plastics are environmentally friendly. However, typical shortcomings include lack of water resistance and poor mechanical properties. Nanocomposite manufacturing using pure, highly linear, polysaccharides can overcome such limitations. Cast nanocomposites were fabricated with plant engineered pure amylose (AM), produced in bulk quantity in transgenic barley grain, and cellulose nanofibers (CNF), extracted from agrowaste sugar beet pulp. Morphology, crystallinity, chemical heterogeneity, mechanics, dynamic mechanical, gas and water permeability, and contact angle of the films were investigated. Blending CNF into the AM matrix significantly enhanced the crystallinity, mechanical properties and permeability, whereas glycerol increased elongation at break, mainly by plasticizing the AM. There was significant phase separation between AM and CNF. Dynamic plasticizing and anti-plasticizing effects of both CNF and glycerol were demonstrated by NMR demonstrating high molecular order, but also non-crystalline, and evenly distributed 20 nm-sized glycerol domains. This study demonstrates a new lead in functional polysaccharide-based bioplastic systems.
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http://dx.doi.org/10.1016/j.carbpol.2020.117277DOI Listing
February 2021

Genome Wide Identification and Comparative Analysis of the Serpin Gene Family in Brachypodium and Barley.

Plants (Basel) 2020 Oct 26;9(11). Epub 2020 Oct 26.

Department of Plant and Environmental Sciences, Faculty of Sciences, University of Copenhagen, 1871 Frederiksberg C, Denmark.

Serpins (serine protease inhibitors) constitute one of the largest and most widely distributed superfamilies of protease inhibitors and have been identified in nearly all organisms. To gain significant insights, a comprehensive in silico analysis of the serpin gene family was carried out in the model plant for temperate grasses and barley using bioinformatic tools at the genome level for the first time. We identified a total of 27 BdSRPs and 25 HvSRP genes in and barley, respectively, showing an unexpectedly high gene number in these model plants. Gene structure, conserved motifs and phylogenetic comparisons of serpin genes supported the role of duplication events in the expansion and evolution of serpin gene family. Further, purifying selection pressure was found to be a main driving force in the evolution of serpin genes. Genome synteny analysis indicated that BdSRP genes were present in syntenic regions of barley, rice, sorghum and maize, suggesting that they evolved before the divergence of these species from common ancestor. The distinct expression pattern in specific tissues further suggested a specialization of functions during development and in plant defense. These results suggest that the LR serpins (serpins with Leu-Arg residues at P2-P1') identified here can be utilized as candidates for exploitation in disease resistance, pest control and preventing stress-induced cell death. Additionally, serpins were identified that could lead to further research aimed at validating and functionally characterizing the role of potential serpin genes from other plants.
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http://dx.doi.org/10.3390/plants9111439DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7692276PMC
October 2020

Response to: Assessment of frailty in geriatric patients: let's keep it simple.

Eur Geriatr Med 2020 10 28;11(5):887. Epub 2020 Aug 28.

Department of Geriatrics, Aarhus University Hospital, Aarhus, Denmark.

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http://dx.doi.org/10.1007/s41999-020-00387-yDOI Listing
October 2020

Golgi-localized exo-β1,3-galactosidases involved in cell expansion and root growth in .

J Biol Chem 2020 07 3;295(31):10581-10592. Epub 2020 Jun 3.

Department of Forest Genetics and Plant Physiology, Umeå Plant Science Centre, Swedish University of Agricultural Sciences, Umeå, Sweden

Plant arabinogalactan proteins (AGPs) are a diverse group of cell surface- and wall-associated glycoproteins. Functionally important AGP glycans are synthesized in the Golgi apparatus, but the relationships among their glycosylation levels, processing, and functionalities are poorly understood. Here, we report the identification and functional characterization of two Golgi-localized exo-β-1,3-galactosidases from the glycosyl hydrolase 43 (GH43) family in GH43 loss-of-function mutants exhibited root cell expansion defects in sugar-containing growth media. This root phenotype was associated with an increase in the extent of AGP cell wall association, as demonstrated by Yariv phenylglycoside dye quantification and comprehensive microarray polymer profiling of sequentially extracted cell walls. Characterization of recombinant GH43 variants revealed that the exo-β-1,3-galactosidase activity of GH43 enzymes is hindered by β-1,6 branches on β-1,3-galactans. In line with this steric hindrance, the recombinant GH43 variants did not release galactose from cell wall-extracted glycoproteins or AGP-rich gum arabic. These results indicate that the lack of exo-β-1,3-galactosidase activity alters cell wall extensibility in roots, a phenotype that could be explained by the involvement of galactosidases in AGP glycan biosynthesis.
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http://dx.doi.org/10.1074/jbc.RA120.013878DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7397118PMC
July 2020

Frailty is associated with hospital readmission in geriatric patients: a prognostic study.

Eur Geriatr Med 2020 10 28;11(5):783-792. Epub 2020 May 28.

Department of Geriatrics, Aarhus University Hospital, Palle Juul-Jensens Boulevard 99, Building, 8200, Aarhus N, Denmark.

Purpose: Targeting health care interventions requires valid measurements when predicting unplanned hospital readmission. The Multidimensional Prognostic Index (MPI) based on Comprehensive Geriatric Assessment (CGA) enables the prediction of mortality and length of stay (LOS) in older hospitalized patients. Our aim was to validate if the MPI as a frailty tool could predict unplanned hospital readmission in geriatric patients.

Methods: This prognostic study was conducted in geriatric wards. The target population was 65 + -year-old patients hospitalized with acute illness. The MPI tool is derived from eight CGA domains by an interdisciplinary team: social aspects, number of drugs, activities of daily living (ADL), instrumental-ADL, cognitive status, severity of morbidity, risk of developing pressure sores, and nutritional status. Patients assessed were categorized into three groups: non-frail (MPI-1), moderate frail (MPI-2) or severe frail (MPI-3). Primary outcome was 30-day unplanned readmission and secondary LOS and 90-day mortality.

Results: In total 1467 patients were included from January 1, 2018, to October 1, 2019. Mean age was 84.2 years (± 7.4) and 59% were women. 15.7% were readmitted. Hazard ratio (HR) for readmission in the MPI-2 group (n = 635) was 2.57; 95% confidence interval (CI) 1.25-5.29 (p = 0.01), and 2.60; 95% CI 1.27-5.33 (p = 0.009) in the MPI-3 group (n = 711) compared to the MPI-1 group (n = 121). MPI was a predictor of LOS and mortality.

Conclusion: Using the MPI tool to identify the frail and non-frail patients is applicable to predict unplanned hospital readmission in geriatric patients. The MPI is superior to the prognostic value of each single domain. MPI will be of great value to health professionals' decision-making.
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http://dx.doi.org/10.1007/s41999-020-00335-wDOI Listing
October 2020

Phenolic cross-links: building and de-constructing the plant cell wall.

Nat Prod Rep 2020 07 23;37(7):919-961. Epub 2020 Jan 23.

Department of Plant and Environmental Sciences, University of Copenhagen, Denmark.

Covering: Up to 2019Phenolic cross-links and phenolic inter-unit linkages result from the oxidative coupling of two hydroxycinnamates or two molecules of tyrosine. Free dimers of hydroxycinnamates, lignans, play important roles in plant defence. Cross-linking of bound phenolics in the plant cell wall affects cell expansion, wall strength, digestibility, degradability, and pathogen resistance. Cross-links mediated by phenolic substituents are particularly important as they confer strength to the wall via the formation of new covalent bonds, and by excluding water from it. Four biopolymer classes are known to be involved in the formation of phenolic cross-links: lignins, extensins, glucuronoarabinoxylans, and side-chains of rhamnogalacturonan-I. Lignins and extensins are ubiquitous in streptophytes whereas aromatic substituents on xylan and pectic side-chains are commonly assumed to be particular features of Poales sensu lato and core Caryophyllales, respectively. Cross-linking of phenolic moieties proceeds via radical formation, is catalyzed by peroxidases and laccases, and involves monolignols, tyrosine in extensins, and ferulate esters on xylan and pectin. Ferulate substituents, on xylan in particular, are thought to be nucleation points for lignin polymerization and are, therefore, of paramount importance to wall architecture in grasses and for the development of technology for wall disassembly, e.g. for the use of grass biomass for production of 2 generation biofuels. This review summarizes current knowledge on the intra- and extracellular acylation of polysaccharides, and inter- and intra-molecular cross-linking of different constituents. Enzyme mediated lignan in vitro synthesis for pharmaceutical uses are covered as are industrial exploitation of mutant and transgenic approaches to control cell wall cross-linking.
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http://dx.doi.org/10.1039/c9np00028cDOI Listing
July 2020

Sustainable production of cellulose nanofiber gels and paper from sugar beet waste using enzymatic pre-treatment.

Carbohydr Polym 2020 Feb 9;230:115581. Epub 2019 Nov 9.

Department of Plant and Environmental Sciences, Section for Glycobiology, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark. Electronic address:

Removal of non-cellulosic polymers from vegetable pulp to obtain cellulose nanofibers (CNF) is normally achieved by chemical pre-treatments which requires several washing steps. In the present study, it is demonstrated how incubation of sugar beet pulp at pH 9, followed by treatment with polysaccharide-degrading enzymes and subsequent bleaching can be done in a one-pot procedure to make CNF. The new method consumes 67% less water and removes non-cellulosic polysaccharides with similar efficiency as a chemical method. In addition, CNF produced by the new method contained slightly more pectin and formed gels with 2.7 times higher storage modulus. Nanopapers cast from chemically- and enzymatically produced CNF showed similar mechanical properties. However, without the pH 9 incubation step, the enzymes accessibility to cell-wall polymers was limited resulting in lower gel and paper strengths. In conclusion, the new method offers a sustainable route for producing high quality CNF from sugar beet waste.
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http://dx.doi.org/10.1016/j.carbpol.2019.115581DOI Listing
February 2020

High efficacy full allelic CRISPR/Cas9 gene editing in tetraploid potato.

Sci Rep 2019 11 27;9(1):17715. Epub 2019 Nov 27.

Department of Plant and Environmental Sciences, University of Copenhagen, DK-1871, Frederiksberg C, Denmark.

CRISPR/Cas9 editing efficacies in tetraploid potato were highly improved through the use of endogenous potato U6 promoters. Highly increased editing efficiencies in the Granular Bound Starch Synthase gene at the protoplast level were obtained by replacement of the Arabidopsis U6 promotor, driving expression of the CRISPR component, with endogenous potato U6 promotors. This translated at the ex-plant level into 35% full allelic gene editing. Indel Detection Amplicon Analysis was established as an efficient tool for fast assessment of gene editing in complex genomes, such as potato. Together, this warrants significant reduction of laborious cell culturing, ex-plant regeneration and screening procedures of plants with high complexity genomes.
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http://dx.doi.org/10.1038/s41598-019-54126-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6881354PMC
November 2019

Cassava cell wall characterization and degradation by a multicomponent NSP-targeting enzyme (NSPase).

Sci Rep 2019 07 12;9(1):10150. Epub 2019 Jul 12.

Novozymes A/S, Krogshoejvej 36, 2880, Bagsværd, Denmark.

Cassava (Manihot esculenta Crantz) is considered the third most important source of calories in tropical regions. Up to one third of cassava harvested worldwide is used in livestock production. The focus of this study was to characterize cassava cell wall structure to provide knowledge for a better application of cassava as an energy source in monogastric animal feed. A total of five cassava samples from different feed mills in South East Asia were investigated. On a dry matter basis, the cassava cell walls contained, on average, 640 mg g glucose, 140 mg g galactose, 50 mg g mannose, 80 mg g xylose, 60 mg g arabinose, 10 mg g fucose and 20 mg g rhamnose. RONOZYME VP (DSM Nutritional Products, Switzerland), a non-specific multicomponent non-starch polysaccharide (NSP) degrading enzyme (NSPase) product from Aspergillus aculeatus, solubilized about 10% of cassava NSP content during 4 h incubations at 40 °C and pH 5. There was notable solubilization of polymers containing uronic acids, galactose, arabinose and rhamnose. Immuno-microscopy imaging indicated the solubilization of pectin, galactan and xyloglucan polysaccharides from cassava cell wall. As a consequence, the starch granules became more available to exogenous α-amylase degradation.
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http://dx.doi.org/10.1038/s41598-019-46341-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6626134PMC
July 2019

Improved CRISPR/Cas9 gene editing by fluorescence activated cell sorting of green fluorescence protein tagged protoplasts.

BMC Biotechnol 2019 06 17;19(1):36. Epub 2019 Jun 17.

Copenhagen Center for Glycomics, Department of Molecular and Cellular Medicine and School of Dentistry, Faculty of Health Sciences, University of Copenhagen, DK-2200, Copenhagen N, Denmark.

Background: CRISPR/Cas9 is widely used for precise genetic editing in various organisms. CRISPR/Cas9 editing may in many plants be hampered by the presence of complex and high ploidy genomes and inefficient or poorly controlled delivery of the CRISPR/Cas9 components to gamete cells or cells with regenerative potential. Optimized strategies and methods to overcome these challenges are therefore in demand.

Results: In this study we investigated the feasibility of improving CRISPR/Cas9 editing efficiency by Fluorescence Activated Cell Sorting (FACS) of protoplasts. We used Agrobacterium infiltration in leaves of Nicotiana benthamiana for delivery of viral replicons for high level expression of gRNAs designed to target two loci in the genome, NbPDS and NbRRA, together with the Cas9 nuclease in fusion with the 2A self-splicing sequence and GFP (Cas9-2A-GFP). Protoplasts isolated from the infiltrated leaves were then subjected to FACS for selection of GFP enriched protoplast populations. This procedure resulted in a 3-5 fold (from 20 to 30% in unsorted to more than 80% in sorted) increase in mutation frequencies as evidenced by restriction enzyme analysis and the Indel Detection by Amplicon Analysis, which allows for high throughput profiling and quantification of the generated mutations.

Conclusions: FACS of protoplasts expressing GFP tagged CRISPR/Cas9, delivered through A. tumefaciens leaf infiltration, facilitated clear CRISPR/Cas9 mediated mutation enrichment in selected protoplast populations.
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http://dx.doi.org/10.1186/s12896-019-0530-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6580576PMC
June 2019

Detection of Seasonal Variation in Polysaccharides Using Carbohydrate Detecting Microarrays.

Front Plant Sci 2019 14;10:512. Epub 2019 May 14.

Natural History Museum of Denmark, University of Copenhagen, Copenhagen, Denmark.

gel is a globally popular natural product used for the treatment of skin conditions. Its useful properties are attributed to the presence of bioactive polysaccharides. Nearly 25% of the 600 species in the genus are used locally in traditional medicine, indicating that the bioactive components in may be common across the genus . The complexity of the polysaccharides has hindered development of relevant assays for authentication of products. Carbohydrate detecting microarrays have recently been suggested as a method for profiling polysaccharide composition. The aim of this study was to use carbohydrate detecting microarrays to investigate the seasonal variation in the polysaccharide composition of two medicinal and two non-medicinal species over the course of a year. Microscopy was used to explore where in the cells the bioactive polysaccharides are present and predict their functional role in the cell wall structure. The carbohydrate detecting microarrays analyses showed distinctive differences in the polysaccharide composition between the different species and carbohydrate detecting microarrays therefore has potential as a complementary screening method directly targeting the presence and composition of relevant polysaccharides. The results also show changes in the polysaccharide composition over the year within the investigated species, which may be of importance for commercial growing in optimizing harvest times to obtain higher yield of relevant polysaccharides.
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http://dx.doi.org/10.3389/fpls.2019.00512DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6527838PMC
May 2019

Dynamics of intracellular mannan and cell wall folding in the drought responses of succulent Aloe species.

Plant Cell Environ 2019 08 3;42(8):2458-2471. Epub 2019 Jun 3.

Department of Comparative Plant and Fungal Biology, Royal Botanic Gardens, Kew, Richmond, TW9 3AE, UK.

Plants have evolved a multitude of adaptations to survive extreme conditions. Succulent plants have the capacity to tolerate periodically dry environments, due to their ability to retain water in a specialized tissue, termed hydrenchyma. Cell wall polysaccharides are important components of water storage in hydrenchyma cells. However, the role of the cell wall and its polysaccharide composition in relation to drought resistance of succulent plants are unknown. We investigate the drought response of leaf-succulent Aloe (Asphodelaceae) species using a combination of histological microscopy, quantification of water content, and comprehensive microarray polymer profiling. We observed a previously unreported mode of polysaccharide and cell wall structural dynamics triggered by water shortage. Microscopical analysis of the hydrenchyma cell walls revealed highly regular folding patterns indicative of predetermined cell wall mechanics in the remobilization of stored water and the possible role of homogalacturonan in this process. The in situ distribution of mannans in distinct intracellular compartments during drought, for storage, and apparent upregulation of pectins, imparting flexibility to the cell wall, facilitate elaborate cell wall folding during drought stress. We conclude that cell wall polysaccharide composition plays an important role in water storage and drought response in Aloe.
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http://dx.doi.org/10.1111/pce.13560DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6851777PMC
August 2019

EXPANSIN A1-mediated radial swelling of pericycle cells positions anticlinal cell divisions during lateral root initiation.

Proc Natl Acad Sci U S A 2019 04 3;116(17):8597-8602. Epub 2019 Apr 3.

School of Biosciences, University of Nottingham, Sutton Bonington LE12 5RD, United Kingdom;

In plants, postembryonic formation of new organs helps shape the adult organism. This requires the tight regulation of when and where a new organ is formed and a coordination of the underlying cell divisions. To build a root system, new lateral roots are continuously developing, and this process requires the tight coordination of asymmetric cell division in adjacent pericycle cells. We identified EXPANSIN A1 (EXPA1) as a cell wall modifying enzyme controlling the divisions marking lateral root initiation. Loss of leads to defects in the first asymmetric pericycle cell divisions and the radial swelling of the pericycle during auxin-driven lateral root formation. We conclude that a localized radial expansion of adjacent pericycle cells is required to position the asymmetric cell divisions and generate a core of small daughter cells, which is a prerequisite for lateral root organogenesis.
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http://dx.doi.org/10.1073/pnas.1820882116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6486723PMC
April 2019

Nanofibers Produced from Agro-Industrial Plant Waste Using Entirely Enzymatic Pretreatments.

Biomacromolecules 2019 01 19;20(1):443-453. Epub 2018 Dec 19.

Department of Plant and Environmental Sciences, Section for Glycobiology , University of Copenhagen , Thorvaldsensvej 40 , 1871 Frederiksberg C , Denmark.

Cellulose fibers can be freed from the cell-wall skeleton via high-shear homogenization, to produce cellulose nanofibers (CNF) that can be used, for example, as the reinforcing phase in composite materials. Nanofiber production from agro-industrial byproducts normally involves harsh chemical-pretreatments and high temperatures to remove noncellulosic polysaccharides (20-70% of dry weight). However, this is expensive for large-scale processing and environmentally damaging. An enzyme-only pretreatment to obtain CNF from agro-industrial byproducts (potato and sugar beet) was developed with targeted commercial enzyme mixtures. It is hypothesized that cellulose can be isolated from the biomass, using enzymes only, due to the low lignin content, facilitating greater liberation of CNF via high-shear homogenization. Comprehensive Microarray Polymer Profiling (CoMPP) measured remaining extractable polysaccharides, showing that the enzyme-pretreatment was more successful at removing noncellulosic polysaccharides than alkaline- or acid-hydrolysis alone. While effective alone, the effect of the enzyme-pretreatment was bolstered via combination with a mild high-pH pretreatment. Dynamic rheology was used to estimate the proportion of CNF in resultant suspensions. Enzyme-pretreated suspensions showed 4-fold and 10-fold increases in the storage modulus for potato and sugar beet, respectively, compared to untreated samples. A greener yet facile method for producing CNF from vegetable waste is presented here.
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http://dx.doi.org/10.1021/acs.biomac.8b01435DOI Listing
January 2019

Interspecies cross-feeding orchestrates carbon degradation in the rumen ecosystem.

Nat Microbiol 2018 11 24;3(11):1274-1284. Epub 2018 Oct 24.

Department of Microbiology, The Ohio State University, Columbus, OH, USA.

Because of their agricultural value, there is a great body of research dedicated to understanding the microorganisms responsible for rumen carbon degradation. However, we lack a holistic view of the microbial food web responsible for carbon processing in this ecosystem. Here, we sampled rumen-fistulated moose, allowing access to rumen microbial communities actively degrading woody plant biomass in real time. We resolved 1,193 viral contigs and 77 unique, near-complete microbial metagenome-assembled genomes, many of which lacked previous metabolic insights. Plant-derived metabolites were measured with NMR and carbohydrate microarrays to quantify the carbon nutrient landscape. Network analyses directly linked measured metabolites to expressed proteins from these unique metagenome-assembled genomes, revealing a genome-resolved three-tiered carbohydrate-fuelled trophic system. This provided a glimpse into microbial specialization into functional guilds defined by specific metabolites. To validate our proteomic inferences, the catalytic activity of a polysaccharide utilization locus from a highly connected metabolic hub genome was confirmed using heterologous gene expression. Viral detected proteins and linkages to microbial hosts demonstrated that phage are active controllers of rumen ecosystem function. Our findings elucidate the microbial and viral members, as well as their metabolic interdependencies, that support in situ carbon degradation in the rumen ecosystem.
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http://dx.doi.org/10.1038/s41564-018-0225-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6784887PMC
November 2018

Analyses of Polysaccharides Using Carbohydrate Microarray Profiling.

J AOAC Int 2018 Nov 12;101(6):1720-1728. Epub 2018 Jun 12.

Natural History Museum of Denmark, University of Copenhagen, Faculty of Science, Sølvgade 83S, DK-1307 Copenhagen K, Denmark.

As the popularity of extracts continues to rise, a desire to fully understand the individual polymer components of the leaf mesophyll, their relation to one another, and the effects they have on the human body are increasing. Polysaccharides present in the leaf mesophyll have been identified as the components responsible for the biological activities of and they have been widely studied in the past decades. However, the commonly used methods do not provide the desired platform to conduct large comparative studies of polysaccharide compositions, as most of them require a complete or near-complete fractionation of the polymers. The objective for this study was to assess whether carbohydrate microarrays could be used for the high-throughput analysis of cell wall polysaccharides in aloe leaf mesophyll. The method we chose is known as comprehensive microarray polymer profiling (CoMPP) and combines the high-throughput capacity of microarray technology with the specificity of molecular probes. Preliminary findings showed that CoMPP can successfully be used for high-throughput screening of aloe leaf mesophyll tissue. Seventeen species of Aloe and closely related genera were analyzed, and a clear difference in the polysaccharide compositions of the mesophyll tissues was seen. These preliminary data suggest that the polysaccharides vary between species and that true species of Aloe may differ from segregate genera.
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http://dx.doi.org/10.5740/jaoacint.18-0120DOI Listing
November 2018

Expression analysis of proteinase inhibitor-II under RGLP2 promoter in response to wounding and signaling molecules in transgenic .

3 Biotech 2018 Jan 4;8(1):51. Epub 2018 Jan 4.

1Department of Plant Sciences, Quaid-i-Azam University, Islamabad, 45320 Pakistan.

Proteinase inhibitor-II (PI-II) genes are important defense related genes that play critical regulatory roles in plant growth and development. In the present study, the expression of tomato gene was investigated under the control of a wound-inducible RGLP2 ( root germin like protein 2) promoter in transgenic tobacco plants after wounding, ABA and MeJA applications. Transcript level of target gene in transgenic plants was confirmed by quantitative real time PCR (qPCR). In response to ABA treatment at different concentrations, - gene was strongly induced under RGLP2 promoter at higher concentration (100 μM), while considerable level of target gene expression was observed with MeJA application at 50 μM concentration. Upon wounding, relatively high - gene expression was observed after 36-h treatment. Correspondingly, high activity was detected at 36 h with histochemical assay and microscopic analysis in the vascular regions of leaves, stem and roots in wounded transgenic plants. This inducibility of - gene by wounding, ABA and MeJA indirectly indicates its role in plant defense mechanism against biotic and abiotic stresses. Moreover, it was also suggested that ABA and MeJA dependent signaling pathways are involved in stimulation of - gene. To the best of our knowledge, this is the first report describing the induction of - gene under the regulation of RGLP2 promoter under stress conditions. The results of present research are useful for potential role of - gene to improve stress tolerance in transgenic crops. Thus, efficacy of this gene can potentially be exploited to test the responses of different plants to various environmental stresses.
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http://dx.doi.org/10.1007/s13205-017-1070-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5754274PMC
January 2018

Development of novel monoclonal antibodies against starch and ulvan - implications for antibody production against polysaccharides with limited immunogenicity.

Sci Rep 2017 08 24;7(1):9326. Epub 2017 Aug 24.

School of Agriculture, Food and Rural Development, Newcastle University, NE1 7RU, Newcastle upon Tyne, UK.

Monoclonal antibodies (mAbs) are widely used and powerful research tools, but the generation of mAbs against glycan epitopes is generally more problematic than against proteins. This is especially significant for research on polysaccharide-rich land plants and algae (Viridiplantae). Most antibody production is based on using single antigens, however, there are significant gaps in the current repertoire of mAbs against some glycan targets with low immunogenicity. We approached mAb production in a different way and immunised with a complex mixture of polysaccharides. The multiplexed screening capability of carbohydrate microarrays was then exploited to deconvolute the specificities of individual mAbs. Using this strategy, we generated a set of novel mAbs, including one against starch (INCh1) and one against ulvan (INCh2). These polysaccharides are important storage and structural polymers respectively, but both are generally considered as having limited immunogenicity. INCh1 and INCh2 therefore represent important new molecular probes for Viridiplantae research. Moreover, since the α-(1-4)-glucan epitope recognised by INCh1 is also a component of glycogen, this mAb can also be used in mammalian systems. We describe the detailed characterisation of INCh1 and INCh2, and discuss the potential of a non-directed mass-screening approach for mAb production against some glycan targets.
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http://dx.doi.org/10.1038/s41598-017-04307-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5570955PMC
August 2017

Rhamnogalacturonan-I Based Microcapsules for Targeted Drug Release.

PLoS One 2016 19;11(12):e0168050. Epub 2016 Dec 19.

Department of Plant and Environmental Sciences, University of Copenhagen, Copenhagen, Denmark.

Drug targeting to the colon via the oral administration route for local treatment of e.g. inflammatory bowel disease and colonic cancer has several advantages such as needle-free administration and low infection risk. A new source for delivery is plant-polysaccharide based delivery platforms such as Rhamnogalacturonan-I (RG-I). In the gastro-intestinal tract the RG-I is only degraded by the action of the colonic microflora. For assessment of potential drug delivery properties, RG-I based microcapsules (~1 μm in diameter) were prepared by an interfacial poly-addition reaction. The cross-linked capsules were loaded with a fluorescent dye (model drug). The capsules showed negligible and very little in vitro release when subjected to media simulating gastric and intestinal fluids, respectively. However, upon exposure to a cocktail of commercial RG-I cleaving enzymes, ~ 9 times higher release was observed, demonstrating that the capsules can be opened by enzymatic degradation. The combined results suggest a potential platform for targeted drug delivery in the terminal gastro-intestinal tract.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0168050PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5167381PMC
July 2017

Degradation of lignin β-aryl ether units in Arabidopsis thaliana expressing LigD, LigF and LigG from Sphingomonas paucimobilis SYK-6.

Plant Biotechnol J 2017 05 29;15(5):581-593. Epub 2016 Nov 29.

Section for Plant Glycobiology, Department of Plant Biology and Environmental Sciences, University of Copenhagen, Frederiksberg C, Denmark.

Lignin is a major polymer in the secondary plant cell wall and composed of hydrophobic interlinked hydroxyphenylpropanoid units. The presence of lignin hampers conversion of plant biomass into biofuels; plants with modified lignin are therefore being investigated for increased digestibility. The bacterium Sphingomonas paucimobilis produces lignin-degrading enzymes including LigD, LigF and LigG involved in cleaving the most abundant lignin interunit linkage, the β-aryl ether bond. In this study, we expressed the LigD, LigF and LigG (LigDFG) genes in Arabidopsis thaliana to introduce postlignification modifications into the lignin structure. The three enzymes were targeted to the secretory pathway. Phenolic metabolite profiling and 2D HSQC NMR of the transgenic lines showed an increase in oxidized guaiacyl and syringyl units without concomitant increase in oxidized β-aryl ether units, showing lignin bond cleavage. Saccharification yield increased significantly in transgenic lines expressing LigDFG, showing the applicability of our approach. Additional new information on substrate specificity of the LigDFG enzymes is also provided.
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http://dx.doi.org/10.1111/pbi.12655DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5399005PMC
May 2017

Pectin nanocoating of titanium implant surfaces - an experimental study in rabbits.

Clin Oral Implants Res 2017 Mar 14;28(3):298-307. Epub 2016 Mar 14.

Institute of Odontology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen N, Denmark.

Introduction: A major determinant of successful osseointegration of endosseous implants is the surface of the implant, which influences the cellular response of the surrounding tissues. A new strategy to improve osseointegration and bone healing is biochemical stimulation by surface nanocoatings that may increase adhesion of bone proteins, and bone cells at the implant surface. Nanocoating with pectins, plant cell wall-derived polysaccharides, is frequently done using rhamnogalacturonan-I (RG-I).

Aim: The aim of the study was to evaluate the effect of nanocoating titanium implants with plant cell wall-derived rhamnogalacturonan-I, on bone healing and osseointegration.

Material And Methods: Machined titanium implants were coated with three modifications of rhamnogalacturonan-I (RG-I). Chemical and physical surface properties were examined before insertion of nanocoated implants (n = 96) into the left and right tibia of rabbits. Machined titanium implants without RG-I nanocoating were used as controls (n = 32). Total number of 128 implants was placed in tibias of 16 rabbits. Fluorochrome bone labels, calcein green and alizarin red S were given intravenously after 9 and 12 days, respectively. The bone response to the nanocoated implants was analyzed qualitatively and quantitatively after 2, 4, 6, and 8 weeks of healing using light microscopy and histomorphometric methods.

Results: The RG-I coating influenced the surface chemical composition; wettability and roughness, making the surface more hydrophilic without any major effect on surface micro roughness compared to control implant surfaces. The different modifications of pectin RG-I did not significantly enhance bone healing and osseointegration analyzed after 2, 4, 6, and 8 weeks of healing compared to control implants. Although the qualitative analyses of the fluorochromes indicated a higher activity of bone formation in the mineralization front at the early stage, after 9 and 12 days at the RG-I nanocoated implants compared to the control implants although no significant quantitative difference was demonstrated.

Conclusion: The present study showed that nanocoating of titanium implants with pectin RG-Is did not significantly enhance bone healing and osseointegration when placed in rabbit tibia bone.
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http://dx.doi.org/10.1111/clr.12798DOI Listing
March 2017

Penium margaritaceum as a model organism for cell wall analysis of expanding plant cells.

Methods Mol Biol 2015 ;1242:1-21

Department of Plant and Environmental Sciences, Faculty ofScience, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Copenhagen, Denmark.

The growth of a plant cell encompasses a complex set of subcellular components interacting in a highly coordinated fashion. Ultimately, these activities create specific cell wall structural domains that regulate the prime force of expansion, internally generated turgor pressure. The precise organization of the polymeric networks of the cell wall around the protoplast also contributes to the direction of growth, the shape of the cell, and the proper positioning of the cell in a tissue. In essence, plant cell expansion represents the foundation of development. Most studies of plant cell expansion have focused primarily upon late divergent multicellular land plants and specialized cell types (e.g., pollen tubes, root hairs). Here, we describe a unicellular green alga, Penium margaritaceum (Penium), which can serve as a valuable model organism for understanding cell expansion and the underlying mechanics of the cell wall in a single plant cell.
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http://dx.doi.org/10.1007/978-1-4939-1902-4_1DOI Listing
July 2015

Osteoblastic response to pectin nanocoating on titanium surfaces.

Mater Sci Eng C Mater Biol Appl 2014 Oct 7;43:117-25. Epub 2014 Jul 7.

Institute of Odontology, Faculty of Health and Medical Sciences, University of Copenhagen, Nørre Allé 20, 2200 Copenhagen N, Denmark.

Osseointegration of titanium implants can be improved by organic and inorganic nanocoating of the surface. The aim of our study was to evaluate the effect of organic nanocoating of titanium surface with unmodified and modified pectin Rhamnogalacturonan-Is (RG-Is) isolated from potato and apple with respect to surface properties and osteogenic response in osteoblastic cells. Nanocoatings on titanium surfaces were evaluated by scanning electron microscopy, contact angle measurements, atomic force microscopy, and X-ray photoelectron spectroscopy. The effect of coated RG-Is on cell adhesion, cell viability, bone matrix formation and mineralization was tested using SaOS-2 cells. Nanocoating with pectin RG-Is affected surface properties and in consequence changed the environment for cellular response. The cells cultured on surfaces coated with RG-Is from potato with high content of linear 1.4-linked galactose produced higher level of mineralized matrix compared with control surfaces and surfaces coated with RG-I with low content of linear 1.4-linked galactose. The study showed that the pectin RG-Is nanocoating not only changed chemical and physical titanium surface properties, but also specific coating with RG-Is containing high amount of galactan increased mineralized matrix formation of osteoblastic cells in vitro.
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http://dx.doi.org/10.1016/j.msec.2014.06.028DOI Listing
October 2014

Pectic arabinan side chains are essential for pollen cell wall integrity during pollen development.

Plant Biotechnol J 2014 May 16;12(4):492-502. Epub 2014 Jan 16.

Wageningen UR Plant Breeding, Wageningen University and Research Centre, Wageningen, the Netherlands.

Pectin is a complex polysaccharide and an integral part of the primary plant cell wall and middle lamella, contributing to cell wall mechanical strength and cell adhesion. To understand the structure-function relationships of pectin in the cell wall, a set of transgenic potato lines with altered pectin composition was analysed. The expression of genes encoding enzymes involved in pectin acetylation, degradation of the rhamnogalacturonan backbone and type and length of neutral side chains, arabinan and galactan in particular, has been altered. Upon crossing of different transgenic lines, some transgenes were not transmitted to the next generation when these lines were used as a pollen donor, suggesting male sterility. Viability of mature pollen was severely decreased in potato lines with reduced pectic arabinan, but not in lines with altered galactan side chains. Anthers and pollen of different developmental stages were microscopically examined to study the phenotype in more detail. Scanning electron microscopy of flowers showed collapsed pollen grains in mature anthers and in earlier stages cytoplasmic protrusions at the site of the of kin pore, eventually leading to bursting of the pollen grain and leaking of the cytoplasm. This phenomenon is only observed after the microspores are released and the tapetum starts to degenerate. Timing of the phenotype indicates a role for pectic arabinan side chains during remodelling of the cell wall when the pollen grain is maturing and dehydrating.
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http://dx.doi.org/10.1111/pbi.12156DOI Listing
May 2014

The structurally effect of surface coated rhamnogalacturonan I on response of the osteoblast-like cell line SaOS-2.

J Biomed Mater Res A 2014 Jun 30;102(6):1961-71. Epub 2013 Jul 30.

Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Thorvaldsensvej 40, Frederiksberg C 1871, Denmark; Copenhagen Center for Glycomics, Institute for Cellular and Molecular Medicine, Faculty of Health and Medicine Sciences, University of Copenhagen, Blegdamsvej 3 B, Copenhagen N 2200, Denmark.

Osseointegration is important when implants are inserted into the bone and can be improved by biochemical surface coating of the implant. In this paper enzymatically modified rhamnogalacturonan I (RG-I) from apple and lupin was used for biochemical coating of aminated surfaces and the importance of the quality of RG-I, the nature of the binding, the fine structure of RG-I, and its effect on SaOS-2 cell line cultured on coated surfaces was investigated. SaOS-2 cells are osteoblast-like cells and a well-established in vitro model of bone-matrix forming osteoblasts. Purification by gel filtration could remove small fragments of galacturonic acid (GalA) and binding studies showed that the purity of the RG-I molecules was important for the quality of the coating. The structure of RG-I and osteoblast-like cells' viability were positively correlated so that high content of 1,4-linked galactose (Gal) and a low content of arabinose in the RG-I molecules favored cell viability. These results indicate that coating of implants with RG-I affect osseointegration positively.
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http://dx.doi.org/10.1002/jbm.a.34868DOI Listing
June 2014

Enzyme affinity to cell types in wheat straw (Triticum aestivum L.) before and after hydrothermal pretreatment.

Biotechnol Biofuels 2013 Apr 16;6(1):54. Epub 2013 Apr 16.

Forest and Landscape, University of Copenhagen, Rolighedsvej 23, Frederiksberg C DK-1958, Denmark.

Background: Wheat straw used for bioethanol production varies in enzymatic digestibility according to chemical structure and composition of cell walls and tissues. In this work, the two biologically different wheat straw organs, leaves and stems, are described together with the effects of hydrothermal pretreatment on chemical composition, tissue structure, enzyme adhesion and digestion. To highlight the importance of inherent cell wall characteristics and the diverse effects of mechanical disruption and biochemical degradation, separate leaves and stems were pretreated on lab-scale and their tissue structures maintained mostly intact for image analysis. Finally, samples were enzymatically hydrolysed to correlate digestibility to chemical composition, removal of polymers, tissue composition and disruption, particle size and enzyme adhesion as a result of pretreatment and wax removal. For comparison, industrially pretreated wheat straw from Inbicon A/S was included in all the experiments.

Results: Within the same range of pretreatment severities, industrial pretreatment resulted in most hemicellulose and epicuticular wax/cutin removal compared to lab-scale pretreated leaves and stems but also in most re-deposition of lignin on the surface. Tissues were furthermore degraded from tissues into individual cells while lab-scale pretreated samples were structurally almost intact. In both raw leaves and stems, endoglucanase and exoglucanase adhered most to parenchyma cells; after pretreatment, to epidermal cells in all the samples. Despite heavy tissue disruption, industrially pretreated samples were not as susceptible to enzymatic digestion as lab-scale pretreated leaves while lab-scale pretreated stems were the least digestible.

Conclusions: Despite preferential enzyme adhesion to epidermal cells after hydrothermal pretreatment, our results suggest that the single most important factor determining wheat straw digestibility is the fraction of parenchyma cells rather than effective tissue disruption.
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http://dx.doi.org/10.1186/1754-6834-6-54DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3650653PMC
April 2013

Toward stable genetic engineering of human O-glycosylation in plants.

Plant Physiol 2012 Sep 12;160(1):450-63. Epub 2012 Jul 12.

Department of Molecular Biology and Genetics, Faculty of Science and Technology, Aarhus University, Flakkebjerg, 4200 Slagelse, Denmark.

Glycosylation is the most abundant and complex posttranslational modification to be considered for recombinant production of therapeutic proteins. Mucin-type (N-acetylgalactosamine [GalNAc]-type) O-glycosylation is found in eumetazoan cells but absent in plants and yeast, making these cell types an obvious choice for de novo engineering of this O-glycosylation pathway. We previously showed that transient implementation of O-glycosylation capacity in plants requires introduction of the synthesis of the donor substrate UDP-GalNAc and one or more polypeptide GalNAc-transferases for incorporating GalNAc residues into proteins. Here, we have stably engineered O-glycosylation capacity in two plant cell systems, soil-grown Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum) Bright Yellow-2 suspension culture cells. Efficient GalNAc O-glycosylation of two stably coexpressed substrate O-glycoproteins was obtained, but a high degree of proline hydroxylation and hydroxyproline-linked arabinosides, on a mucin (MUC1)-derived substrate, was also observed. Addition of the prolyl 4-hydroxylase inhibitor 2,2-dipyridyl, however, effectively suppressed proline hydroxylation and arabinosylation of MUC1 in Bright Yellow-2 cells. In summary, stably engineered mammalian type O-glycosylation was established in transgenic plants, demonstrating that plants may serve as host cells for the production of recombinant O-glycoproteins. However, the present stable implementation further strengthens the notion that elimination of endogenous posttranslational modifications may be needed for the production of protein therapeutics.
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http://dx.doi.org/10.1104/pp.112.198200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3440218PMC
September 2012