Prakash M. Dey

Plant cell-walls

Publisher Summary This chapter provides an overview of plant cell-walls. The cell wall is an envelope that encases the plant cell. The wall must be rigid enough to give the plant strength and form, and yet, if necessary, it must yield freely to facilitate growth. The network of cell walls, where adjoining cells have a wall in common, provides in a plant the structural framework analogous to both the skin and the bones of an animal. In some plants, especially those having woody tissues, the strength of this cell-wall network is prodigious. However, despite their apparently tough sheathing, the cells of the growing regions of a plant are able to extend to many times their initial length. The cell wall lies outside the plasma membrane, which defines the boundaries of the cell itself. The wall is freely permeable to most molecules, but the membrane exhibits selective permeability tending to concentrate certain dissolved molecules and ions inside the cell. The presence of such charged components as acidic polysaccharides within the wall imparts ion-exchange properties to the wall. The chapter discusses the concepts related to the types of cell-wall polysaccharides and elaborates the methods used in the elucidation of primary-wall structure. It also presents an overview of cell-wall glycoproteins.

Biochemistry of Plant Galactomannans

Publisher Summary Plant galactomannans are reserve polysaccharides composed of linear chains of (1→4)-linked β-D-mannopyranosyl residues having single stubs of α-D-galactopyranosyl groups joined by (1→6)-linkages along the chain. These polysaccharides are widely used in industry especially in food, pharmaceuticals, cosmetics, paper products, paints and plasters, well-drilling and mining, and explosives and fire-fighting. The distribution of galactomannans in the plant kingdom is limited. The rich sources are the members of the family Leguminoseae. In context to the biosynthesis of plant galactomannans, it is concluded that D-galactose is stored in the seeds in the form of galactomannan, by random attachment of D-galactosyl groups to a main-chain of D-mannan. Seed galactomannans exhibits double physiological function. Firstly, they retain water by solvation and thereby prevent complete drying of the seeds, which can cause protein denaturation— in particular—the denaturation of those enzymes essential for seed germination. Secondly, the galactomannans serve as food reserves for the germinating seeds.

Dose Responses for Colletotrichum lindemuthianum Elicitor-mediated Enzyme Induction in French Bean Cell Suspension Cultures

The induction of L-phenylalanine ammonialyase (PAL, EC 4.3.1.5) and flavanone synthase in French bean cell suspension cultures in response to heat-released elicitor from cell walls of the phytopathogenic fungus Colletotrichum lindemuthianum is highly dependent upon elicitor concentration. The elicitor dose-response curve for PAL induction shows two maxima at around 17.5 and 50 μg elicitor carbohydrate per ml culture, whereas the flavanone synthase response shows one maximum at around 100 μg ml-1. The PAL response is independent of the elicitor concentration present during the lag phase of enzyme induction; if the initial elicitor concentration is increased after 2 h by addition of extra elicitor, or decreased by dilution of the cultures, the dose response curves obtained reflect the concentration of elicitor present at the time of harvest. PAL induction is not prevented by addition of methyl sugar derivatives to the cultures; α-methyl-D-glucoside, itself a weak elicitor of PAL activity, elicits a multiphasic PAL response when increasing concentrations are added in the presence of Colletotrichum elicitor. Eight fractions with different monosaccharide compositions, obtained from the crude elicitor by gel-filtration, each elicit different dose-responses for PAL induction; the response to unfractionated elicitor is not the sum of the response to the isolated fractions. There is no correlation between the ability of the fractions to induce PAL in the cultures and their ability to act as elicitors of isoflavonoid phytoalexin accumulation in bean hypocotyls.

Postharvest Changes in Fruit Cell Wall

Publisher Summary From a physiological point of view, a fruit can be defined as the structural entity resulting from the development of the tissue that supports the ovule. The plant cell consists of cytoplasm surrounded by a cell wall. Each cell is connected to the adjacent cells by a pectin-rich middle lamella. Fruits are generally harvested at the mature stage when growth has ceased, and full development may be achieved independent of the parent plant with negligible impairment to quality. Fruits and their products constitute a commercially significant food commodity. The primary cell wall constituents can be divided into pectic polysaccharides (34%), hemicellulose (24%), cellulose (23%), and hydroxy proline-rich glycoprotein (19%). Pectic polysaccharides are generally considered to be those portions of cell walls that can be extracted by a variety of mild methods such as hot water, ammonium oxalate solution, weak acids, chelating agents, and endopolygalacturonase. The way in which the various components of the cell wall are linked together is largely not known. This is due mainly to the limited amount of information currently available on their structures. From a physiological point of view, a fruit results from the development of the tissue that supports the ovule of a plant. It is certain that tissue softening during the process of fruit ripening is related to breakdown of the organized structure of primary cell wall. The polygalacturonase-catalyzed degradation of pectic rhamnogalacturonan resulting in the dissolution of the middle lamella is probably the major contributor to tissue softening during fruit ripening.

Purification and Partial Characterization of Tomato Extensin Peroxidase

Early plant defense response is characterized by elevation of activity of peroxidases and enhanced insolubilization of hydroxyproline-rich glycoproteins, such as extensin, in the cell wall. The insolubilization process (cross-linking between soluble extensin precursor molecules) is catalyzed by extensin peroxidases. We have ionically eluted extensin peroxidases from intact water-washed suspension-cultured tomato (hybrid of Lycopersicon esculentum Mill. and Lycopersicon peruvianum L. [Mill.]) cells and purified them to homogeneity by molecular sieve and cation-exchange chromatography. Four ionic forms of peroxidase (PI,PII,EPIII, and EPIV) were resolved; only the latter two cross-linked tomato soluble extensin. The molecular weight (34,000–37,000), amino acid composition, and isoelectric point (9.0) of the extensin peroxidases were determined. Substrate specificities of the enzymes were investigated: soluble extensin and potato lectin (a hydroxyproline-rich glycoprotein with a domain that strongly resembles extensin) were cross-linked by only two forms of the enzyme, whereas bovine serum albumin, aldolase, insulin, a number of other marker proteins, and proteins eluted from tomato cells (except extensin) could not be cross-linked. We have also isolated a yeast elicitor that enhances total peroxidase activity and extensin insolubilization within 1 h of challenge in cultured cells of tomato. A highly sensitive enzyme-linked immunosorbent assay technique using polyclonal antiserum raised against soluble tomato extensin was used to demonstrate extensin insolubilization in vivo. A tomato cell-wall peroxidase that cross-links extensin has been purified and may have a role in plant defense.

Biochemistry of α-D-Galactosidic Linkages in the Plant Kingdom

Publisher Summary This chapter provides an overview of the biochemistry of α- D -Galactosidic linkages in the plant kingdom. Sucrose is a disaccharide that is most widespread amongst higher plants; its concentration varies from species to species. The metabolism of this disaccharide is well documented. Sucrose is formed as a major product of photosynthesis in higher plants and is generally the main form of translocate from leaves to other organs. It is a major carbohydrate-storage material that provides a ready source of D -glucose and D -fructose for the liberation of energy. Sucrose is also an important precursor for the synthesis of D -glucosyl esters of nucleoside diphosphates, and thus it takes part in the biosynthesis of complex oligosaccharides and polysaccharides. Combination of D -galactose with the D -glucosyl or D -fructosyl group of the sucrose molecule gives rise to a number of oligosaccharides; these are, generally, α- D -galactosides. The chapter discusses in detail about α- D -galactosides of sucrose. It also describes the α- D -galactosides of polyols and presents an overview of α- D -galactopyranosyl-specific lectins.

Purification and properties of chalcone isomerase from cell suspension cultures of Phaseolus vulgaris

Abstract Chalcone isomerase (EC 5.5.1.6) from cell suspension cultures of Phaseolus vulgaris has been purified about 400-fold. The molecular weight, as estimated by gel-filtration and SDS-polyacrylamide gel electrophoresis, is approx. 28 000. No isoenzymic forms are observed. The enzyme, which appears to require no cofactors, catalyses the isomerisation of both 6′-hydroxy and 6′-deoxy chalcones to the corresponding flavones. Likewise, a range of both 5-hydroxy and 5-deoxy flavonoids and isoflavonoids act as competitive inhibitors. The most potent inhibitors include the naturally occurring antimicrobial comcpounds kievitone ( K i 9.2 μ M) and coumestrol ( K i 2.5 μ M). The kinetics of the isomerisation of 2′,4,4′-trihydroxychalcone to the flavanone liquiritigenin have been investigated at a range of pH values. The pH optimum was around 8.0 and K m changed with pH in a manner consistent with control by groups which ionise with p K a values of 7.05 and 8.7 respectively. At pH 8.0, the energy of activation was 17.56 kJ/mol in the range 25–40°C. The role of the enzyme in the induced accumulation of flavonoid/isoflavonoid derivatives inthe Frech bean in discussed.

Studies on the distribution of α-galactosidases in seeds

Abstract Dormant seeds from various higher plant species have been examined for multiple forms of α-galactosidase. The properties of corresponding forms from Pisum sativum and Vicia faba have been compared. The high molecular from (I) from P. sativum has been purified 2770-fold.

Post-harvest changes in Mangifera indica mesocarp cell walls and cytoplasmic polysaccharides

Abstract During ripening, mango mesocarp cell walls undergo degradation with the net loss of arabinose, galactose and galacturonic acid. Hot water fractions of the cell walls from unripe fruits were rich in galactose and arabinose and contained only 7% galacturonic acid in comparison with those from unripe fruits which contained 90% uronic acid: little change occurred in the alkali-soluble (hemicellulose) fraction during ripening. The ripening-associated changes in the cold water-soluble cytoplasmic polysaccharides in the mesocarp were also examined. As the mesocarp softened these increased in amount and bound uronic acid increased three-fold. Gel-filtration and ion-exchange chromatography were used to examine these cytoplasmic polysaccharides. Their average M r decreased on ripening and most of the fractions were complex with respect to monosaccharide composition. However, polysaccharides which are essentially an arabinoxylan and a galacturonan appeared to be present in the unripe and ripe tissue, respectively.

Purification of extensin from cell walls of tomato (hybrid of Lycopersicon esculentum and L. peruvianum) cells in suspension culture

Extensin, a hydroxyproline-rich glycoprotein comprising substantial amounts of β-l-arabinose-hydroxyproline glycosidic linkages is believed to be insolubilized in the cell wall during host-pathogen interaction by a peroxidase/hydroperoxide-mediated cross-linking process. Both extensin precursor and extensin peroxidase were ionically eluted from intact water-washed tomato (hybrid) of Lycopersicon esculentum Mill. and L. peruvianum L. (Mill.) cells in suspension cultures and purified to homogeneity by a rapid and simple procedure under mild and non-destructive experimental conditions. The molecular weight of native extensin precursor was estimated to be greater than 240–300 kDa by Superose-12 gel-filtration chromatography. Extensin monomers have previously been designated a molecular weight of approximately 80 kDa. Our results indicate that salt-eluted extensin precursor is not monomeric. Agarose-gel electrophoresis, Superose-12-gel-filtration, extensin-peroxidase-catalysed cross-linking, Mono-S ion-exchange fast protein liquid chromatography (FPLC), and peptide-sequencing data confirmed the homogeneity of the extensin preparation. Evidence that the purified protein was extensin is attributed to the presence of the putative sequence motif — Ser (Hyp)4 — within the N-terminal end of the protein. Treatment of extensin with trifluoroacetic acid demonstrated that arabinose was the principal carbohydrate. The amino-acid composition of the purified extensin was similar to those reported in the literature. The cross-linking of extensin in vitro upon incubation with extensin peroxidase and exogenous H2O2 was characteristic of other reported extensins. Furthermore, Mono-S ion-exchange FPLC of native extensin precursor resolved it into two isoforms, A (90%) and B (10%). The amino-acid compositions of extensin A and extensin B were found to be similar to each other and both extensins were cross-linked in vitro by extensin peroxidase.