Radiometric and spectrophotometric in vitro assays of glycosyltransferases involved in plant cell wall carbohydrate biosynthesis

2012 ◽  
Vol 7 (9) ◽  
pp. 1634-1650 ◽  
Author(s):  
Christian Brown ◽  
Felicia Leijon ◽  
Vincent Bulone
Keyword(s):  
Cell Wall ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
In Vitro Assays

scholarly journals The Role of Brachypodium distachyon Wall-Associated Kinases (WAKs) in Cell Expansion and Stress Responses

Cells ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 2478
Author(s):  
Xingwen Wu ◽  
Antony Bacic ◽  
Kim L. Johnson ◽  
John Humphries
Keyword(s):  
Cell Wall ◽  
Stress Response ◽  
Plant Cell ◽  
Stress Responses ◽  
Cell Expansion ◽  
Plant Cell Wall ◽  
Brachypodium Distachyon ◽  
Critical Role ◽  
Cell Integrity

The plant cell wall plays a critical role in signaling responses to environmental and developmental cues, acting as both the sensing interface and regulator of plant cell integrity. Wall-associated kinases (WAKs) are plant receptor-like kinases located at the wall—plasma membrane—cytoplasmic interface and implicated in cell wall integrity sensing. WAKs in Arabidopsis thaliana have been shown to bind pectins in different forms under various conditions, such as oligogalacturonides (OG)s in stress response, and native pectin during cell expansion. The mechanism(s) WAKs use for sensing in grasses, which contain relatively low amounts of pectin, remains unclear. WAK genes from the model monocot plant, Brachypodium distachyon were identified. Expression profiling during early seedling development and in response to sodium salicylate and salt treatment was undertaken to identify WAKs involved in cell expansion and response to external stimuli. The BdWAK2 gene displayed increased expression during cell expansion and stress response, in addition to playing a potential role in the hypersensitive response. In vitro binding assays with various forms of commercial polysaccharides (pectins, xylans, and mixed-linkage glucans) and wall-extracted fractions (pectic/hemicellulosic/cellulosic) from both Arabidopsis and Brachypodium leaf tissues provided new insights into the binding properties of BdWAK2 and other candidate BdWAKs in grasses. The BdWAKs displayed a specificity for the acidic pectins with similar binding characteristics to the AtWAKs.

scholarly journals Fermentation and subsequent disposition of 14C-labelled plant cell wall material in the rat

1993 ◽  
Vol 69 (1) ◽  
pp. 189-197 ◽  
Author(s):  
D. F. Gray ◽  
M. A. Eastwood ◽  
W. G. Brydon ◽  
S. C. Fry
Keyword(s):  
Cell Wall ◽  
Gastrointestinal Tract ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Spinacia Oleracea ◽  
Wall Material ◽  
Fermentation Products ◽  
Bacterial Fermentation ◽  
Cell Wall Preparation

A 14C-Iabelled plant cell wall preparation (I4C-PCW) produced from spinach (Spinacia oleracea L.) cell culture exhibits uniform labelling of the major polysaccharide groups (%): pectins 53, hemicellulose 13, cellulose 21, starch 3. This 14C-PCW preparation has been used in rat studies as a marker for plant cell wall metabolism. Metabolism of the 14C-PCW occurred largely over the first 24 h. This was due to fermentation in the caecum. The pectic fraction of the plant cell walls was degraded completely in the rat gastrointestinal tract, but some [14C-]cellulose was still detected after 24 h in the colon. Of the 14C,22% was recovered in the host liver, adipose tissue and skin, 26% excreted as 14CO2 and up to 18%was excreted in the faeces. There was no urinary excretion of 14C. In vitro fermentation using a caecal inocuium showed reduced 14CO2 production, 12% compared with 26% in the intact rat. 14C-PCW is auseful marker to investigate the fate of plant cell wall materials in the gastrointestinal tract. These studies show both bacterial fermentation of the 14C-PCW and host metabolism of the 14C-labelled fermentation products.

scholarly journals Immunocytochemical Localization of HrpA and HrpZ Supports a Role for the Hrp Pilus in the Transfer of Effector Proteins from Pseudomonas syringae pv. tomato Across the Host Plant Cell Wall

2001 ◽  
Vol 14 (3) ◽  
pp. 394-404 ◽  
Author(s):  
Ian R. Brown ◽  
John W. Mansfield ◽  
Suvi Taira ◽  
Elina Roine ◽  
Martin Romantschuk
Keyword(s):  
Cell Wall ◽  
Pseudomonas Syringae ◽  
Plant Cell ◽  
Type Iii Secretion ◽  
Plant Cell Wall ◽  
Immunogold Labeling ◽  
Effector Proteins ◽  
Basal Bodies ◽  
Type Iii

The Hrp pilus, composed of HrpA subunits, is an essential component of the type III secretion system in Pseudomonas syringae. We used electron microscopy (EM) and immunocytochemistry to examine production of the pilus in vitro from P. syringae pv. tomato strain DC3000 grown under hrp-inducing conditions on EM grids. Pili, when labeled with antibodies to HrpA, developed rapidly in a nonpolar manner shortly after the detection of the hrpA transcript and extended up to 5 μm into surrounding media. Structures at the base of the pilus were clearly differentiated from the basal bodies of flagella. The HrpZ protein, also secreted via the type III system, was found by immunogold labeling to be associated with the pilus in vitro. Accumulation and secretion of HrpA and HrpZ were also examined quantitatively after the inoculation of wild-type DC3000 and hrpA and hrpZ mutants into leaves of Arabidopsis thaliana. The functional pilus crossed the plant cell wall to generate tracks of immunogold labeling for HrpA and HrpZ. Mutants that produced HrpA but did not assemble pili were nonpathogenic, did not secrete HrpA protein, and were compromised for the accumulation of HrpZ. A model is proposed in which the rapidly elongating Hrp pilus acts as a moving conveyor, facilitating transfer of effector proteins from bacteria to the plant cytoplasm across the formidable barrier of the plant cell wall.

scholarly journals A mixed-linkage (1,3;1,4)-β-D-glucan specific hydrolase mediates dark-triggered degradation of this plant cell wall polysaccharide

2021 ◽  
Author(s):  
Florian J Kraemer ◽  
China Lunde ◽  
Moritz Koch ◽  
Benjamin M Kuhn ◽  
Clemens Ruehl ◽  
...  
Keyword(s):  
Cell Wall ◽  
Plant Cell ◽  
Large Scale ◽  
Plant Cell Wall ◽  
Causative Mutation ◽  
Grass Species ◽  
Calorie Intake ◽  
Cell Wall Polysaccharide ◽  
Transcript Accumulation

Abstract The presence of mixed-linkage (1,3;1,4)-β-D-glucan (MLG) in plant cell walls is a key feature of grass species such as cereals, the main source of calorie intake for humans and cattle. Accumulation of this polysaccharide involves the coordinated regulation of biosynthetic and metabolic machineries. While several components of the MLG biosynthesis machinery have been identified in diverse plant species, degradation of MLG is poorly understood. In this study, we performed a large-scale forward genetic screen for maize (Zea mays) mutants with altered cell wall polysaccharide structural properties. As a result, we identified a maize mutant with increased MLG content in several tissues, including adult leaves and senesced organs, where only trace amounts of MLG are usually detected. The causative mutation was found in the GRMZM2G137535 gene, encoding a GH17 licheninase as demonstrated by an in vitro activity assay of the heterologously expressed protein. In addition, maize plants overexpressing GRMZM2G137535 exhibit a 90% reduction in MLG content, indicating that the protein is not only required, but its expression is sufficient to degrade MLG. Accordingly, the mutant was named MLG hydrolase 1 (mlgh1). mlgh1 plants show increased saccharification yields upon enzymatic digestion. Stacking mlgh1 with lignin-deficient mutations results in synergistic increases in saccharification. Time profiling experiments indicate that wall MLG content is modulated during day/night cycles, inversely associated with MLGH1 transcript accumulation. This cycling is absent in the mlgh1 mutant, suggesting that the mechanism involved requires MLG degradation, which may in turn regulate MLGH1 gene expression.

scholarly journals Enzyme-assisted in vivo polymerisation of conjugated oligomer based conductors

2020 ◽  
Vol 8 (19) ◽  
pp. 4221-4227 ◽  
Author(s):  
Gwennaël Dufil ◽  
Daniela Parker ◽  
Jennifer Y. Gerasimov ◽  
Thuc-Quyen Nguyen ◽  
Magnus Berggren ◽  
...  
Keyword(s):  
Cell Wall ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Plant Structure

The conjugated oligomer ETE-S is enzymatically polymerized in vitro, in the presence of peroxidase and H2O2. This polymerization route occurs also in the plant cell wall where ETE-S polymerizes and forms conductors along the plant structure.

scholarly journals A specific fungal transcription factor controls effector gene expression and orchestrates the establishment of the necrotrophic pathogen lifestyle on wheat

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Darcy A. B. Jones ◽  
Evan John ◽  
Kasia Rybak ◽  
Huyen T. T. Phan ◽  
Karam B. Singh ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Wall ◽  
Transcription Factor ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Cell Wall Degradation ◽  
Necrotrophic Pathogen ◽  
Effector Gene ◽  
Plant Cell Wall Degradation

Abstract The fungus Parastagonospora nodorum infects wheat through the use of necrotrophic effector (NE) proteins that cause host-specific tissue necrosis. The Zn2Cys6 transcription factor PnPf2 positively regulates NE gene expression and is required for virulence on wheat. Little is known about other downstream targets of PnPf2. We compared the transcriptomes of the P. nodorum wildtype and a strain deleted in PnPf2 (pf2-69) during in vitro growth and host infection to further elucidate targets of PnPf2 signalling. Gene ontology enrichment analysis of the differentially expressed (DE) genes revealed that genes associated with plant cell wall degradation and proteolysis were enriched in down-regulated DE gene sets in pf2-69 compared to SN15. In contrast, genes associated with redox control, nutrient and ion transport were up-regulated in the mutant. Further analysis of the DE gene set revealed that PnPf2 positively regulates twelve genes that encode effector-like proteins. Two of these genes encode proteins with homology to previously characterised effectors in other fungal phytopathogens. In addition to modulating effector gene expression, PnPf2 may play a broader role in the establishment of a necrotrophic lifestyle by orchestrating the expression of genes associated with plant cell wall degradation and nutrient assimilation.

scholarly journals Confirmation of Cage Effect and Prebiotic Production Potential of a β-Mannanase, with SBM as Substrate Using Microscopy and Wet Chemistry

2021 ◽  
Vol 13 (2) ◽  
pp. 23
Author(s):  
L. M. Gomez-Osorio ◽  
Hwa Gyun Oh ◽  
Jung Jin Lee
Keyword(s):  
Cell Wall ◽  
Cell Walls ◽  
Plant Cell Wall ◽  
Degree Of Polymerization ◽  
In Vitro Assays ◽  
Wall Structure ◽  
Specific Substrate ◽  
Mannan Oligosaccharides

In vitro assays were carried out to investigate the solubilization of cell walls and generation of mannan oligosaccharides of a b-mannanase-containing commercial product on SBM. Using commercial dosages of the b-mannanase (500 g per ton of feed) cell wall degradation of mannan in SBM cell walls was visualized and an increase in reducing ends (0.12±0.02 mg/mL) and the generation of mannan oligosaccharides of degree of polymerization 2 and 4 (22.9±3.2 mg/L and 398.8±25.4 mg/L) were also measured using HPLC. Mannan, which is H-bonded to cellulose and xyloglucan, was solubilized using a single monocomponent enzyme, allowing for visualization of the disintegration of the entire SBM cell wall structure. This work is the first of its kind using strictly commercial dosage levels of enzyme for evaluating efficacy of the same microscopically. These data confirm the hypothesis that there most likely is a need for only a single relevant NSP enzyme targeting its specific substrate, independent of the concentration of the latter within the complex polysaccharide matrix in the plant cell wall to experience the beneficial effects of the enzyme both in vitro and in vivo. An analogy to compare our data would be destruction of the foundation (mannan) of a building or a bridge (soybean cell wall) which would inevitably lead to dismantling or demolition the entire building or bridge.

scholarly journals The Phytophthora infestans Haustorium Is a Site for Secretion of Diverse Classes of Infection-Associated Proteins

mBio ◽  
2018 ◽  
Vol 9 (4) ◽  
Author(s):  
Shumei Wang ◽  
Lydia Welsh ◽  
Peter Thorpe ◽  
Stephen C. Whisson ◽  
Petra C. Boevink ◽  
...  
Keyword(s):  
Cell Wall ◽  
Phytophthora Infestans ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Extracellular Proteins ◽  
Host Cells ◽  
Cysteine Protease Inhibitor ◽  
Plant Interactions ◽  
Content Type

ABSTRACT The oomycete potato blight pathogen Phytophthora infestans secretes a diverse set of proteins to manipulate host plant immunity. However, there is limited knowledge about how and where they are secreted during infection. Here we used the endoplasmic reticulum (ER)-to-Golgi secretion pathway inhibitor brefeldin A (BFA) in combination with liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) to identify extracellular proteins from P. infestans that were conventionally secreted from in vitro-cultured hyphae. We identified 19 proteins with predicted signal peptides that potentially influence plant interactions for which secretion was attenuated by BFA. In addition to inhibition by the apoplastic effector EPIC1, a cysteine protease inhibitor, we show that secretion of the cell wall-degrading pectinesterase enzyme PE1 and the microbe-associated molecular pattern (MAMP)-like elicitin INF4 was inhibited by BFA in vitro and in planta, demonstrating that these proteins are secreted by the conventional, Golgi-mediated pathway. For comparison, secretion of a cytoplasmic RXLR (Arg-[any amino acid]-Leu-Arg) effector, Pi22926, was not inhibited by BFA. During infection, whereas INF4 accumulated outside the plant cell, RXLR effector Pi22926 entered the plant cell and accumulated in the nucleus. The P. infestans effectors, the PE1 enzyme, and INF4 were all secreted from haustoria, pathogen structures that penetrate the plant cell wall to form an intimate interaction with the host plasma membrane. Our findings show the haustorium to be a major site of both conventional and nonconventional secretion of proteins with diverse functions during infection. IMPORTANCE There are many different classes of proteins secreted from Phytophthora infestans that may influence or facilitate infection. Elucidating where and how they are secreted during infection is an important step toward developing methods to control their delivery processes. We used an inhibitor of conventional secretion to identify the following different classes of infection-associated extracellular proteins: cell wall-degrading and cell wall-modifying enzymes, microbe-associated molecular pattern-like proteins that may elicit immune responses, and apoplastic effectors that are predicted to suppress immunity. In contrast, secretion of a cytoplasmic effector that is translocated into host cells is nonconventional, as it is insensitive to inhibitor treatment. This evidence further supports the finding that proteins that are active in the apoplast and effector proteins that are active in the host cytoplasm are differentially secreted by P. infestans. Critically, it demonstrates that a disease-specific developmental structure, the haustorium, is a major secretion site for diverse protein classes during infection.

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