Analysis of Partially Methyl-Esterified Galacturonic Acid Oligomers by High-Performance Anion-Exchange Chromatography and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

Daas, Piet; Arisz, Peter W.; Schols, Henk A.; Ruiter, G.A. de; Voragen, A.G.J.

doi:10.1006/abio.1997.2554

Cited by 90 publications

(56 citation statements)

References 27 publications

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“…The matrix suspension was prepared by dissolving 9 mg of 2,5-dihydroxybenzoic acid and 3 mg of isocarbostyril in 1 ml of a water-acetonitrile mixture (7:3). The mass spectrometer was calibrated externally with a polygalacturonic acid endo-PG digest (7).…”

Section: Methodsmentioning

confidence: 99%

Endo-Xylogalacturonan Hydrolase, a Novel Pectinolytic Enzyme

Vlugt-Bergmans

Meeuwsen²,

Voragen³

et al. 2000

Appl Environ Microbiol

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We screened an Aspergillus tubingensis expression library constructed in the yeast Kluyveromyces lactis for xylogalacturonan-hydrolyzing activity in microwell plates by using a bicinchoninic acid assay. This assay detects reducing carbohydrate groups when they are released from a carbohydrate by enzymatic activity. Two K. lactis recombinants exhibiting xylogalacturonan-hydrolyzing activity were found among the 3,400 colonies tested. The cDNA insert of these recombinants encoded a 406-amino-acid protein, designated XghA, which was encoded by a single-copy gene, xghA. A multiple-sequence alignment revealed that XghA was similar to both polygalacturonases (PGs) and rhamnogalacturonases. A detailed examination of conserved regions in the sequences of these enzymes revealed that XghA resembled PGs more. High-performance liquid chromatography and matrix-assisted laser desorption ionization-time of flight mass spectrometry of the products of degradation of xylogalacturonan and saponified modified hairy regions of apple pectin by XghA demonstrated that this enzyme uses an endo type of mechanism. XghA activity appeared to be specific for a xylose-substituted galacturonic acid backbone.Pectin occurs as constituent of higher-plant cell walls, where it is embedded in the cellulose fibrils. The composition of pectin is different in different plant species and also depends on the age and maturity of the plant part. Most pectin polymers consist of smooth homogalacturonan regions and ramified hairy regions. The smooth regions consist of a linear homogalacturonan backbone. The hairy regions, as identified in apples (28), consist of three different subunits; subunit I is xylogalacturonan (xga) (a galacturonan backbone heavily substituted with xylose), subunit II is a short section of a rhamnogalacturonan backbone that has many relatively long arabinan, galactan, and/or arabinogalactan side chains (the hairs), and subunit III is rhamnogalacturonan composed of alternating rhamnose and galacturonic acid residues.

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Section: Methodsmentioning

confidence: 99%

Endo-Xylogalacturonan Hydrolase, a Novel Pectinolytic Enzyme

Vlugt-Bergmans

Meeuwsen²,

Voragen³

et al. 2000

Appl Environ Microbiol

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“…The negatively charged sugars bind to the column material and elute through competitive binding with an increasing salt gradient [135]. As an alternative, pectin oligosaccharides can be analyzed at pH 5 to retain information about methyl esters distribution over the backbone [136]. After elution, sugars are often detected by a pulsed ampherometric detector [135].…”

Section: Analytical Approachesmentioning

confidence: 99%

Pectin, a versatile polysaccharide present in plant cell walls

et al. 2009

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Pectin or pectic substances are collective names for a group of closely associated polysaccharides present in plant cell walls where they contribute to complex physiological processes like cell growth and cell differentiation and so determine the integrity and rigidity of plant tissue. They also play an important role in the defence mechanisms against plant pathogens and wounding. As constituents of plant cell walls and due to their anionic nature, pectic polysaccharides are considered to be involved in the regulation of ion transport, the porosity of the walls and in this way in the control of the permeability of the walls for enzymes. They also determine the water holding capacity. The amount and composition of pectic molecules in fruits and vegetables and other plant produce strongly determine quality parameters of fresh and processed food products. Pectin is also extracted from suitable agro-by-products like citrus peel and apple pomace and used in the food industry as natural ingredients for their gelling, thickening, and stabilizing properties. Some pectins gain more and more interest for their health modulating activities. Endogenous as well as exogenous enzymes play an important role in determining the pectic structures present in plant tissue, food products, or ingredients at a given time. In this paper functional and structural characteristics of pectin are described with special emphasis on the structural elements making up the pectin molecule, their interconnections and present models which envisage the accommodation of all structural elements in a macromolecule. Attention is also given to analytical methods to study the pectin structure including the use of enzymes as analytical tools. PectinPectin is one of the major plant cell wall components and probably the most complex macromolecule in nature, as it can be composed out of as many as 17 different monosaccharides containing more than 20 different linkages [1][2][3]. Plant functionality of pectinIn a plant, pectin is present in the middle lamella, primary cell and secondary walls and is deposited in the early stages of growth during cell expansion [4]. Its functionality to a plant is quite divers. First, pectin plays an important role in the formation of higher plant cell walls [5], which lend strength and support to a plant and yet are very dynamic structures [4]. In general, the polymeric composition of primary cell walls in dicotyledonous plants consists of approximately 35% pectin, 30% cellulose, 30% hemicellulose, and 5% protein [5]. Grasses contain 2-10% pectin and wood tissue ca 5%. In cell walls of some fruits and vegetables, the pectin content can be substantially higher and the protein content lower [6]. Second, pectin influences various cell wall properties such as porosity, surface charge, pH, and ion balance and therefore is of importance to the ion transport in the cell wall [7]. Furthermore, pectin oligosaccharides are known to activate plant defense

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“…In particular, in the G n (m,a) fragment, ''G n '' denotes the number of GalUA residues in the backbone, and ''m'' and ''a'' indicate the number of methylesterified and of acetylated residues, respectively. The main ions detected by MALDI-TOF MS analysis were assigned to (M1Na 1 ) 1 adducts on the basis of their M r and literature data (Daas et al, 1998; Table III). Differences in MALDI-TOF mass spectra were observed in both highly expressing AtPMEI-1 and AtPMEI-2 lines in comparison with wild type (Fig.…”

Section: Overexpression Of Atpmei In Arabidopsis Affects the Degree Omentioning

confidence: 99%

Overexpression of Pectin Methylesterase Inhibitors in Arabidopsis Restricts Fungal Infection by Botrytis cinerea

Lionetti

Raiola

Camardella³

et al. 2007

Plant Physiology

313

306

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(M.P.) Pectin, one of the main components of plant cell wall, is secreted in a highly methylesterified form and is demethylesterified in muro by pectin methylesterase (PME). The action of PME is important in plant development and defense and makes pectin susceptible to hydrolysis by enzymes such as endopolygalacturonases. Regulation of PME activity by specific protein inhibitors (PMEIs) can, therefore, play a role in plant development as well as in defense by influencing the susceptibility of the wall to microbial endopolygalacturonases. To test this hypothesis, we have constitutively expressed the genes AtPMEI-1 and AtPMEI-2 in Arabidopsis (Arabidopsis thaliana) and targeted the proteins into the apoplast. The overexpression of the inhibitors resulted in a decrease of PME activity in transgenic plants, and two PME isoforms were identified that interacted with both inhibitors. While the content of uronic acids in transformed plants was not significantly different from that of wild type, the degree of pectin methylesterification was increased by about 16%. Moreover, differences in the fine structure of pectins of transformed plants were observed by enzymatic fingerprinting. Transformed plants showed a slight but significant increase in root length and were more resistant to the necrotrophic fungus Botrytis cinerea. The reduced symptoms caused by the fungus on transgenic plants were related to its impaired ability to grow on methylesterified pectins.Pectin is a structurally complex polysaccharide that accounts for nearly 35% of the dicot and nongraminaceous monocot primary cell wall. A main component of pectin is homogalacturonan (HGA) consisting of a backbone of 1,4-linked a-D-GalUA units, with variable amounts of methylester in the C 6 position. Pectins are secreted into the cell wall in a highly methylesterified form and, soon thereafter, are deesterified in muro by pectin methylesterase (PME; Brummell and Harpster, 2001;Willats et al., 2001). Demethylesterification produces free carboxyl groups and modifies the pH and charge of the wall, allowing the aggregation of polyuronides into a calcium-linked gel structure and increasing the wall firmness (Willats et al., 2001). In addition, the action of PMEs makes HGA susceptible to degradation by hydrolases such as endopolygalacturonases (endoPGs), contributing to the softening of the cell wall (Brummell and Harpster, 2001;Wakabayashi et al., 2003).Plant PMEs are involved in important physiological processes such as microsporogenesis, pollen growth, pollen separation, seed germination, root development, polarity of leaf growth, stem elongation, fruit ripening, and loss of tissue integrity

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Analysis of Partially Methyl-Esterified Galacturonic Acid Oligomers by High-Performance Anion-Exchange Chromatography and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

Cited by 90 publications

References 27 publications

Endo-Xylogalacturonan Hydrolase, a Novel Pectinolytic Enzyme

Endo-Xylogalacturonan Hydrolase, a Novel Pectinolytic Enzyme

Pectin, a versatile polysaccharide present in plant cell walls

Overexpression of Pectin Methylesterase Inhibitors in Arabidopsis Restricts Fungal Infection by Botrytis cinerea

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