STUDIES ON RHAMNOGALACTURONASE IN FRUIT

Rhamnogalacturonase (RGase) is a new fungal enzyme which degrades the highly branched regions of apple fruit cell wall pectin by cleaving the glycosyl linkage between rhamnosyl and galacturonosyl residues (Schols et al., 1990. Carhohydr. Res. 206:105.). This enzyme, if present in fruit, could play a significant role in fruit softening. Partial purification of RGase was accomplished from a fungal enzyme preparation (Pectinex Ultra SP-L, NOVO Ferment) produced from Aspergillus niger. The crude enzyme hydrolyzed chelator-soluble pectin from red ripe tomato fruit. Methylation linkage analysis of the product suggested that an increase in terminal-rhamnosyl residues accompanied pectin hydrolysis, indicative of RGase activity. Cross-linked alginate, hydroxyapatite, and DEAE-Sephadex chromatography were used to partially purify RGase. Polygalacturonase was efficiently removed using the alginate column. Crude pectin obtained from mature-green tomato fruit cell wall by extracting with 0.5 M imidazole buffer (pH 7) and 50 mM Na-carbonate was incubated with pure polygalacturonase and the residue hydrolyzed with 0.1 N trifluoroacetic acid. This modified pectin was used as a substrate to investigate the presence of RGase in tomato and other fruit.

Download Full-text

Altered cell wall disassembly during ripening of Cnr tomato fruit: implications for cell adhesion and fruit softening

Planta ◽

10.1007/s00425-002-0753-1 ◽

2002 ◽

Vol 215 (3) ◽

pp. 440-447 ◽

Cited By ~ 41

Author(s):

Caroline Orfila ◽

Miranda Huisman ◽

William Willats ◽

Gert-Jan van Alebeek ◽

Henk Schols ◽

...

Keyword(s):

Cell Wall ◽

Cell Adhesion ◽

Tomato Fruit ◽

Fruit Softening ◽

Altered Cell ◽

Cell Wall Disassembly

Download Full-text

(472) Yeast Expressed Tomato β-Galactosidases 1, 4, and 5 Have Activity against Synthetic and Plant-derived Cell Wall Substrates

HortScience ◽

10.21273/hortsci.40.4.1092b ◽

2005 ◽

Vol 40 (4) ◽

pp. 1092B-1092 ◽

Cited By ~ 2

Author(s):

Megumi Ishimaru ◽

David L. Smith ◽

Kenneth C. Gross

Keyword(s):

Cell Wall ◽

Tomato Fruit ◽

Wall Material ◽

Wall Structure ◽

Fruit Softening ◽

Pectic Polysaccharides ◽

Wide Range ◽

Fruit Pericarp ◽

Galactosyl Residues

Fruit softening occurs by several mechanisms, including modifications of cell wall structure by wall degrading enzymes. The most prominent change in tomato fruit pericarp wall composition is the loss of galactosyl residues throughout development and especially during ripening. In order to understand the role of galactosyl turnover in fruit softening, we successfully produced three recombinant tomato β-galactosidase/exo-galactanase (TBG) fusion proteins in yeast. TBG1, 4 and 5 enzyme properties and substrate specificities were assessed. Optimum pH of TBG1, 4 and 5 was 5.0, 4.0, and 4.5 and optimum temperature was 40∼50, 40, and 40 °C, respectively. The K ms for TBG1, 4 and 5 were 7.99, 0.09, and 2.42 mm, respectively, using p-nitrophenyl-β-D-galactopyranoside as substrate. Using synthetic and plant-derived substrates, TBG1 and 5 released galactosyl residues from 1 → 4 linkages. TBG4 released galactosyl residues from a wide range of plant-derived oligosaccharides and polysaccharides. Using tomato fruit cell wall material, TBG1, TBG4 and TBG5 released galactosyl residues from a variety of fruit stages and cell wall fractions. TBG4 released the most galactosyl residues from the ASP fraction and especially the ASP fraction from fruit at the turning stage. Interestingly, even though walls from Turning fruit stage contain less total galactosyl residues than at the Mature Green stage, TBG4 released 3–4 fold more galactose from the CSP and ASP fractions from Turning fruit. These results suggest that changes in structure of wall pectic polysaccharides leading up to the Turning stage may cause the wall to become more susceptible to hydrolysis by the TBG4 product.

Download Full-text

657 Genetic Determinants and Control of Fruit Softening

HortScience ◽

10.21273/hortsci.35.3.511d ◽

2000 ◽

Vol 35 (3) ◽

pp. 511D-511

Author(s):

Alan B. Bennett

Keyword(s):

Gene Expression ◽

Cell Wall ◽

Shelf Life ◽

Fruit Quality ◽

General Model ◽

Early Stage ◽

Tomato Fruit ◽

Fruit Softening ◽

Expansin Gene ◽

Cell Wall Disassembly

Fruit softening is integral to the ripening process. It is an important component of fruit quality, but also initiates deterioration and is a limiting determinant of shelf-life. Intensive research has attempted to elucidate the biochemical and genetic control of fruit softening with the goal of controlling this process as a means to enhance both fruit quality and shelf-life. Current models of fruit softening focus on cell wall disassembly as the major biochemical event regulating fruit softening. Examination of the sequence of cell wall disassembly in ripening Charentais melon fruit suggested that softening could be divided into two distinct phases. The early stage of fruit softening was associated with the regulated disassembly of xyloglucan polymers and the later softening that accompanies over-ripe deterioration was associated with pectin depolymerization. Characterization of cell wall changes in other fruit, including tomato, suggest that this may represent a general model of sequential cell wall disassembly in ripening fruit. Interestingly, the early events of xyloglucan disassembly were not associated with the activation or expression of xyloclucan hydrolases but were associated with the expression of a ripening-regulated expansin gene. Analysis of transgenic tomato fruit with suppressed expansin gene expression or with suppressed polygalacturonase gene expression supports a general model of sequential disassembly of xyloglucan and pectin that control the early and late phases of fruit softening, respectively.

Download Full-text

Pectinesterase, Polygalacturonase, and β -galactosidase during Softening of Ethylene-treated Kiwifruit

HortScience ◽

10.21273/hortsci.27.8.900 ◽

1992 ◽

Vol 27 (8) ◽

pp. 900-902 ◽

Cited By ~ 34

Author(s):

Teresa F. Wegrzyn ◽

Elspeth A. MacRae

Keyword(s):

Cell Wall ◽

Actinidia Deliciosa ◽

Fruit Softening ◽

Ethylene Treatment ◽

Fungal Enzyme ◽

Low Levels

The activities of several cell wall-associated enzymes of the outer pericarp were assayed during softening of kiwifruit [Actinidia deliciosa (A. Chev.) C.F. Liang et A.R. Ferguson var. deliciosa cv. Hayward] treated with ethylene. The activity of polygalacturonase (EC 3.2.1.15) increased slightly during fruit softening, while β-galactosidase (EC 3.2.1.23) activity remained constant. Salt-extracted pectinesterase (EC 3.1.1.11) activity increased during ethylene treatment, then dropped rapidly to low levels as fruit softened. Residual pectinesterase activity, extracted after digestion of the cell wall pellet with a fungal enzyme mix, decreased on softening. The rapid softening of kiwifruit in response to ethylene treatment may be initiated by an induction of pectinesterase activity, causing increased de-esterification of cell wall pectins, followed by degradation of solubilized pectin.

Download Full-text

Stimulation of ethylene production by a cell wall component from mature green tomato fruit

Physiologia Plantarum ◽

10.1034/j.1399-3054.1990.800402.x ◽

1990 ◽

Vol 80 (4) ◽

pp. 500-506 ◽

Cited By ~ 1

Author(s):

Cindy B. S. Tong ◽

Kenneth C. Gross

Keyword(s):

Cell Wall ◽

Ethylene Production ◽

Tomato Fruit ◽

Cell Wall Component ◽

A Cell ◽

Stimulation Of

Download Full-text

Potent Chitin Synthase Inhibitors from Plants

Current Bioactive Compounds ◽

10.2174/1573407213666180719145831 ◽

2020 ◽

Vol 16 (1) ◽

pp. 58-63

Author(s):

Amrutha Vijayakumar ◽

Ajith Madhavan ◽

Chinchu Bose ◽

Pandurangan Nanjan ◽

Sindhu S. Kokkal ◽

...

Keyword(s):

Cell Wall ◽

Candida Albicans ◽

Caenorhabditis Elegans ◽

Aspergillus Niger ◽

Small Molecules ◽

Tip Growth ◽

Chitin Synthase ◽

Chitin Synthesis ◽

Hyphal Tip Growth ◽

Hyphal Tip

Background: Chitin is the main component of fungal, protozoan and helminth cell wall. They help to maintain the structural and functional characteristics of these organisms. The chitin wall is dynamic and is repaired, rearranged and synthesized as the cells develop. Active synthesis can be noticed during cytokinesis, laying of primary septum, maintenance of lateral cell wall integrity and hyphal tip growth. Chitin synthesis involves coordinated action of two enzymes namely, chitin synthase (that lays new cell wall) and chitinase (that removes the older ones). Since chitin synthase is conserved in different eukaryotic microorganisms that can be a ‘soft target’ for inhibition with small molecules. When chitin synthase is inhibited, it leads to the loss of viability of cells owing to the self- disruption of the cell wall by existing chitinase. Methods: In the described study, small molecules from plant sources were screened for their ability to interfere with hyphal tip growth, by employing Hyphal Tip Burst assay (HTB). Aspergillus niger was used as the model organism. The specific role of these small molecules in interfering with chitin synthesis was established with an in-vitro method. The enzyme required was isolated from Aspergillus niger and its activity was deduced through a novel method involving non-radioactively labelled substrate. The activity of the potential lead molecules were also checked against Candida albicans and Caenorhabditis elegans. The latter was adopted as a surrogate for the pathogenic helminths as it shares similarity with regard to cell wall structure and biochemistry. Moreover, it is widely studied and the methodologies are well established. Results: Out of the 11 compounds and extracts screened, 8 were found to be prospective. They were also found to be effective against Candida albicans and Caenorhabditis elegans. Conclusion: Purified Methyl Ethyl Ketone (MEK) Fraction1 (F1) of Coconut (Cocos nucifera) Shell Extract (COSE) was found to be more effective against Candida albicans with an IC50 value of 3.04 μg/mL and on L4 stage of Caenorhabditis elegans with an IC50 of 77.8 μg/mL.

Download Full-text