Isolation and Characterization of Secretory Vesicles in Germinated Pollen of Lilium longiflorum

1971 ◽  
Vol 8 (2) ◽  
pp. 331-351 ◽  
Author(s):  
W. J. VAN DER WOUDE ◽  
D. J. MORRÉ ◽  
C. E. BRACKER
Keyword(s):  
Cell Wall ◽  
Secretory Pathway ◽  
Periodic Acid ◽  
Lilium Longiflorum ◽  
Water Soluble ◽  
Hot Water ◽  
Secretory Vesicles ◽  
Vesicle Membrane ◽  
Isolation And Characterization ◽  
Germinated Pollen

Secretory vesicles containing polysaccharide were isolated from germinated pollen of Lilium longiflorum and characterized by biochemical and ultrastructural investigation. Pollen tubes exhibit a secretory pathway in which the vesicles concentrated in the tube apex are produced by the Golgi apparatus and contributed to the cell wall at the apex upon fusion of the vesicle membrane with the plasma membrane. Secretory vesicles were isolated by a method involving the size discrimination of cytoplasmic components using Millipore filters. Cells were disrupted under conditions which minimized membrane vesiculation. Identification was made by electron-microscopic comparison of the periodic acid-silver hexamine (PASH) reactivities of in situ and isolated secretory vesicles. The secretory vesicles contained polysaccharides which were high in galacturonic acid and similar in sugar composition to those of the hot-water-soluble fraction of pollen tube cell wall. A hot-water-insoluble, non-cellulosic glucan was the major component of the cell wall. Less than 7% of the wall was cellulosic. Chitin was absent. Similarities in the ultrastructure and PASH staining of apical secretory vesicles and an amorphous component of the cell wall support a precursor-product relationship between these 2 cell components. Ultrastructural investigations revealed complexes of the endoplasmic reticulum (ER) associated with electron-translucent regions of cytoplasm, suggesting a possible function of the ER in cell wall formation. Additionally, patterns of PASH staining show that changes in polysaccharides occur in secretory vesicles after vesicles have been formed by dictyosomes. Therefore, secretory vesicles may have a role in polysaccharide synthesis as well as in membrane and product transport.

scholarly journals Ultrastructural and biochemical analyses reveal cell wall remodelling in lichen-forming microalgae submitted to cyclic desiccation–rehydration

2019 ◽  
Vol 125 (3) ◽  
pp. 459-469 ◽  
Author(s):  
María González-Hourcade ◽  
Marcia R Braga ◽  
Eva M del Campo ◽  
Carmen Ascaso ◽  
Cristina Patiño ◽  
...  
Keyword(s):  
Cell Wall ◽  
Close Contact ◽  
Freeze Substitution ◽  
Water Soluble ◽  
Hot Water ◽  
Cell Shrinkage ◽  
Distinctive Features ◽  
Biochemical Analyses ◽  
Species Specific ◽  
Environmental Sem

Abstract Background and Aims One of the most distinctive features of desiccation-tolerant plants is their high cell wall (CW) flexibility. Most lichen microalgae can tolerate drastic dehydration–rehydration (D/R) conditions; however, their mechanisms of D/R tolerance are scarcely understood. We tested the hypothesis that D/R-tolerant microalgae would have flexible CWs due to species-specific CW ultrastructure and biochemical composition, which could be remodelled by exposure to cyclic D/R. Methods Two lichen microalgae, Trebouxia sp. TR9 (TR9, adapted to rapid D/R cycles) and Coccomyxa simplex (Csol, adapted to seasonal dry periods) were exposed to no or four cycles of desiccation [25–30 % RH (TR9) or 55–60 % RH (Csol)] and 16 h of rehydration (100 % RH). Low-temperature SEM, environmental SEM and freeze-substitution TEM were employed to visualize structural alterations induced by D/R. In addition, CWs were extracted and sequentially fractionated with hot water and KOH, and the gel permeation profile of polysaccharides was analysed in each fraction. The glycosyl composition and linkage of the main polysaccharides of each CW fraction were analysed by GC–MS. Key Results All ultrastructural analyses consistently showed that desiccation caused progressive cell shrinkage and deformation in both microalgae, which could be rapidly reversed when water availability increased. Notably, the plasma membrane of TR9 and Csol remained in close contact with the deformed CW. Exposure to D/R strongly altered the size distribution of TR9 hot-water-soluble polysaccharides, composed mainly of a β-3-linked rhamnogalactofuranan and Csol KOH-soluble β-glucans. Conclusions Cyclic D/R induces biochemical remodelling of the CW that could increase CW flexibility, allowing regulated shrinkage and expansion of D/R-tolerant microalgae.

Isolation and Characterization of a Polysaccharide Antigen from Propionibacterium acnes Released by a Glycine-Specific Chemical Protein Degradation Procedure

1990 ◽  
Vol 45 (7-8) ◽  
pp. 797-804
Author(s):  
Mary Fran Verostek ◽  
Lee E. Bartholomew ◽  
Peter Weber
Keyword(s):  
Molecular Weight ◽  
Cell Wall ◽  
Propionibacterium Acnes ◽  
Culture Media ◽  
Water Soluble ◽  
High Molecular Weight Fraction ◽  
Mercuric Oxide ◽  
Specific Chemical ◽  
Antigenic Polysaccharide ◽  
Isolation And Characterization

Abstract An acid-labile antigenic polysaccharide has been isolated from both cell walls and culture media of Propionibacterium acnes using a new chemical degradation procedure which liberates protein-bound or associated carbohydrate. Lyophilized cells and culture media were treated with a suspension of mercuric oxide in a solution of alkaline mercuric cyanide for several hours at room temperature liberating water-soluble polysaccharide material. The antigenic polysac- charide was freed of reaction products by alcohol extraction and purified by anion exchange chromatography and gel filtration, resulting in three distinct fractions of acidic polysaccha- rides of apparent molecular weights between 15 - 50 kDa. Sugar analysis showed the polysac- charides to contain fucose, galactose, glucose, mannose, galactosamine. glucosamine, and 2,3- diamino-2.3-dideoxy-D-glucuronic acid. The three fractions also contained amino acids, predominantly glutamic acid, alanine, and glycine, known to be components of P. acnes cell wall peptidoglycan. All three molecular weight fractions reacted with rabbit antisera raised against whole P. acnes cells, with the highest titer for both cell and media-derived polysaccharide material consistently in the high molecular weight fraction. This procedure was also capable of releasing antigenic polysaccharide from tissues of rats administered P. acnes cells or radio- labeled cell wall fragments.

Studies of the structure and chemical composition of the cell walls of Vaucheriaceae and Saprolegniaceae

1963 ◽  
Vol 158 (973) ◽  
pp. 435-445 ◽  
Keyword(s):  
Cell Wall ◽  
Cell Walls ◽  
Insoluble Fraction ◽  
Water Soluble ◽  
Hot Water ◽  
Cellulose Microfibrils ◽  
Wall Material ◽  
Native Cellulose ◽  
A Cell ◽  
Crystalline Component

The cell walls of members of the Vaucheriaceae and Saprolegniaceae have been examined by X-ray analysis and electron microscopy, and their composition determined by hydrolysis and paper partition chromatography of the hydrolysates. Both differences and similarities between the members of these two species examined are found to supplement the comparative morphological and physiological information at present available. Saprolegnia , Achlya , Brevilegnia and Dictyuchus among the Saprolegniaceae possess hot-water soluble polysaccharides containing glucose residues only. This polysaccharide is not crystallographically identical with the polysaccharide found in Vaucheria sessilis with a similar solubility. The members of the Saprolegniaceae contain large amounts of alkali-soluble polysaccharides in contrast with the negligible amount found in V. sessilis . These polysaccharides are only weakly crystalline, but the indications are that the same polysaccharides may occur through­out the Saprolegniaceae. The alkali-insoluble wall material of Vaucheria species consists of highly crystalline native cellulose with large, apparently randomly arranged, microfibrils. The hydrolysate of this material contains ribose, xylose and arabinose in addition to glucose, presumably representing strongly bound pentosans. Native cellulose also occurs in the Saprolegniaceae but only in small proportion. The bulk of the alkali-insoluble fraction in the walls of these fungi appears amorphous in the electron microscope and is only weakly crystalline. It consists of one or m ore substances containing glucose, mannose, ribose and possibly other sugars together with traces of glucosamine. These substances presumably cover the cellulose microfibrils. The total quantity of non-cellulosic polysaccharide in the Saprolegniaceae approaches 85% of the total wall weight in contrast with the situation in Vaucheria where the cellulose alone approaches 90% of the total cell wall. Dichotomosiphon is unique among the organism s studied in this paper, in possessing a cell wall entirely soluble in alkali and composed of approximately equal quantities of glucose and xylose. The crystalline component is aβ-1,3-linked xylan, as already reported for some of the Siphonales (closely related algae) by Frei & Preston.

scholarly journals A Eukaryotic Capsular Polysaccharide Is Synthesized Intracellularly and Secreted via Exocytosis

2006 ◽  
Vol 17 (12) ◽  
pp. 5131-5140 ◽  
Author(s):  
Aki Yoneda ◽  
Tamara L. Doering
Keyword(s):  
Cell Wall ◽  
Cell Membrane ◽  
Virulence Factor ◽  
Secretory Pathway ◽  
Capsular Polysaccharide ◽  
Extracellular Polysaccharides ◽  
Restrictive Temperature ◽  
Secretory Vesicles ◽  
Biosynthetic Pathways ◽  
Major Virulence Factor

Cryptococcus neoformans, which causes fatal infection in immunocompromised individuals, has an elaborate polysaccharide capsule surrounding its cell wall. The cryptococcal capsule is the major virulence factor of this fungal organism, but its biosynthetic pathways are virtually unknown. Extracellular polysaccharides of eukaryotes may be made at the cell membrane or within the secretory pathway. To test these possibilities for cryptococcal capsule synthesis, we generated a secretion mutant in C. neoformans by mutating a Sec4/Rab8 GTPase homolog. At a restrictive temperature, the mutant displayed reduced growth and protein secretion, and accumulated ∼100-nm vesicles in a polarized manner. These vesicles were not endocytic, as shown by their continued accumulation in the absence of polymerized actin, and could be labeled with anti-capsular antibodies as visualized by immunoelectron microscopy. These results indicate that glucuronoxylomannan, the major cryptococcal capsule polysaccharide, is trafficked within post-Golgi secretory vesicles. This strongly supports the conclusion that cryptococcal capsule is synthesized intracellularly and secreted via exocytosis.

scholarly journals Hot Water Treatment Maintains Normal Ripening and Cell Wall Metabolism in Mango (Mangifera indica L.) Fruit

HortScience ◽  
2012 ◽  
Vol 47 (10) ◽  
pp. 1466-1471 ◽  
Author(s):  
Zhengke Zhang ◽  
Zhaoyin Gao ◽  
Min Li ◽  
Meijiao Hu ◽  
Hui Gao ◽  
...  
Keyword(s):  
Cell Wall ◽  
Water Treatment ◽  
Low Temperature ◽  
Mangifera Indica ◽  
Water Soluble ◽  
Hot Water ◽  
Hot Water Treatment ◽  
Cell Wall Metabolism ◽  
Low Temperature Storage ◽  
Temperature Storage

‘Tainong 1’ mango fruit were treated with hot water for 10 minutes at 55 °C and then stored at 5 °C for 3 weeks. After removal from low-temperature storage, the effects of hot water treatment (HWT) on chilling injury (CI), ripening and cell wall metabolism during storage (20 °C, 5 days) were investigated. HWT reduced the CI development of the fruit as manifested by firmer texture, external browning, and fungal lesions. A more rapid ripening process, as indicated by changes in firmness, respiration rate, and ethylene production, occurred in heated fruit after exposure to low temperature as compared with non-heated fruit. At the same time, the cell wall components in heated fruit contained more water-soluble pectin and less 1,2-cyclohexylenedinitrilotetraactic acid (CDTA)-soluble pectin than those in non-heated fruit. HWT also maintained higher polygalacturonase [enzyme classification (EC) 3.2.1.15] and β-galactosidase (EC 3.2.1.23) activities as well as lower pectin methylesterase (EC 3.1.1.11) activity. In general, the changes of ripening and cell wall metabolism parameters in the heated fruit after low-temperature storage exhibited a comparable pattern to that of non-cold-stored fruit.

AN ANALYSIS OF AVENA COLEOPTILE PECTIN FRACTIONS

1965 ◽  
Vol 43 (9) ◽  
pp. 1083-1095 ◽  
Author(s):  
D. James Morré ◽  
Alfred C. Olson
Keyword(s):  
Cell Wall ◽  
Galacturonic Acid ◽  
Water Soluble ◽  
Hot Water ◽  
Plant Tissues ◽  
Cytoplasmic Fraction ◽  
Avena Coleoptile ◽  
Heat Stable ◽  
Heat Stable Protein

Extraction and determination of pectic materials from growing plant tissues is often complicated by overlapping solubilities and lack of specificity of the pectin assay utilized. We find that the hot water soluble, hot versene soluble, and residual uronide components of Avena coleoptile cell wall represent at least three distinct pectin fractions with little or no overlap in solubility. In situations where hexose interference in colorimetric pectin determinations became appreciable, the polyanhydrogalacturonic acid content of the extract was determined by measurement of isolated galacturonic acid released through the specific action of polygalacturonase.A fourth fraction containing pectin-like materials was extracted from whole tissue in cold acetate buffer. This fraction was associated with heat-stable protein. No pectin identified as polyanhydrogalacturonic acid was found in the cytoplasmic fraction by the same techniques used for identifying pectin on cell wall derived fractions.

Isolation and characterization of cell walls from the mesocarp of mature grape berries (Vitis vinifera)

2020 ◽  
Author(s):  
KJ Nunan ◽  
Ian Sims ◽  
A Bacic ◽  
SP Robinson ◽  
GB Fincher
Keyword(s):  
Vitis Vinifera ◽  
Cell Walls ◽  
Vascular Tissue ◽  
Compositional Analysis ◽  
Water Soluble ◽  
Vitis Vinifera L ◽  
Nylon Mesh ◽  
Cytoplasmic Material ◽  
Isolation And Characterization ◽  
Tissue Homogenates

Cell walls have been isolated from the mesocarp of mature grape (Vitis vinifera L.) berries. Tissue homogenates were suspended in 80% (v/v) ethanol to minimise the loss of water-soluble wall components and wet-sieved on nylon mesh to remove cytoplasmic material. The cell wall fragments retained on the sieve were subsequently treated with buffered phenol at pH 7.0, to inactivate any wall-bound enzymes and to dislodge small amounts of cytoplasmic proteins that adhered to the walls. Finally, the wall preparation was washed with chloroform/methanol (1:1, v/v) to remove lipids and dried by solvent exchange. Scanning electron microscopy showed that the wall preparation was essentially free of vascular tissue and adventitious protein of cytoplasmic origin. Compositional analysis showed that the walls consisted of approximately 90% by weight of polysaccharide and less than 10% protein. The protein component of the walls was shown to be rich in arginine and hydroxyproline residues. Cellulose and polygalacturonans were the major constituents, and each accounted for 30-40% by weight of the polysaccharide component of the walls. Substantial varietal differences were observed in the relative abundance of these two polysaccharides. Xyloglucans constituted approximately 10% of the polysaccharide fraction and the remainder was made up of smaller amounts of mannans, heteroxylans, arabinans and galactans.

scholarly journals New methods for confocal imaging of infection threads in crop and model legumes

Plant Methods ◽  
2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Angus E. Rae ◽  
Vivien Rolland ◽  
Rosemary G. White ◽  
Ulrike Mathesius
Keyword(s):  
Cell Wall ◽  
Root Hair ◽  
Periodic Acid ◽  
Confocal Imaging ◽  
Wall Material ◽  
Future Research ◽  
Thin Sections ◽  
New Methods ◽  
Infection Threads ◽  
Imaging Of Infection

Abstract Background The formation of infection threads in the symbiotic infection of rhizobacteria in legumes is a unique, fascinating, and poorly understood process. Infection threads are tubes of cell wall material that transport rhizobacteria from root hair cells to developing nodules in host roots. They form in a type of reverse tip-growth from an inversion of the root hair cell wall, but the mechanism driving this growth is unknown, and the composition of the thread wall remains unclear. High resolution, 3-dimensional imaging of infection threads, and cell wall component specific labelling, would greatly aid in our understanding of the nature and development of these structures. To date, such imaging has not been done, with infection threads typically imaged by GFP-tagged rhizobia within them, or histochemically in thin sections. Results We have developed new methods of imaging infection threads using novel and traditional cell wall fluorescent labels, and laser confocal scanning microscopy. We applied a new Periodic Acid Schiff (PAS) stain using rhodamine-123 to the labelling of whole cleared infected roots of Medicago truncatula; which allowed for imaging of infection threads in greater 3D detail than had previously been achieved. By the combination of the above method and a calcofluor-white counter-stain, we also succeeded in labelling infection threads and plant cell walls separately, and have potentially discovered a way in which the infection thread matrix can be visualized. Conclusions Our methods have made the imaging and study of infection threads more effective and informative, and present exciting new opportunities for future research in the area.

scholarly journals Evaluating polymer interplay after hot water pretreatment to investigate maize stem internode recalcitrance

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Amandine Leroy ◽  
Xavier Falourd ◽  
Loïc Foucat ◽  
Valérie Méchin ◽  
Fabienne Guillon ◽  
...  
Keyword(s):  
Cell Wall ◽  
Negative Impact ◽  
Lignin Content ◽  
Structural Features ◽  
Hot Water ◽  
Condensed State ◽  
Structural Factors ◽  
Water Pretreatment ◽  
Hot Water Pretreatment ◽  
The Impact

Abstract Background Biomass recalcitrance is governed by various molecular and structural factors but the interplay between these multiscale factors remains unclear. In this study, hot water pretreatment (HWP) was applied to maize stem internodes to highlight the impact of the ultrastructure of the polymers and their interactions on the accessibility and recalcitrance of the lignocellulosic biomass. The impact of HWP was analysed at different scales, from the polymer ultrastructure or water mobility to the cell wall organisation by combining complementary compositional, spectral and NMR analyses. Results HWP increased the kinetics and yield of saccharification. Chemical characterisation showed that HWP altered cell wall composition with a loss of hemicelluloses (up to 45% in the 40-min HWP) and of ferulic acid cross-linking associated with lignin enrichment. The lignin structure was also altered (up to 35% reduction in β–O–4 bonds), associated with slight depolymerisation/repolymerisation depending on the length of treatment. The increase in $${T}_{1\rho }^{H}$$ T 1 ρ H , $${T}_{HH}$$ T HH and specific surface area (SSA) showed that the cellulose environment was looser after pretreatment. These changes were linked to the increased accessibility of more constrained water to the cellulose in the 5–15 nm pore size range. Conclusion The loss of hemicelluloses and changes in polymer structural features caused by HWP led to reorganisation of the lignocellulose matrix. These modifications increased the SSA and redistributed the water thereby increasing the accessibility of cellulases and enhancing hydrolysis. Interestingly, lignin content did not have a negative impact on enzymatic hydrolysis but a higher lignin condensed state appeared to promote saccharification. The environment and organisation of lignin is thus more important than its concentration in explaining cellulose accessibility. Elucidating the interactions between polymers is the key to understanding LB recalcitrance and to identifying the best severity conditions to optimise HWP in sustainable biorefineries.

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