Root Slime Research Articles

Behaviour of zoospores and zoospore cysts in relation to root infection by Pythium aphanidermatum

summaryThe Pre‐Penetration Sequence Involving Zoospore Taxis, encystment, Cyst Germination And Germ‐Tube Orientation Was Studied By Video Microscopy When Roots Of Wheat, Cress or Tomato Were Placed In Suspensions of Zoospores of Pythium aphanidermatum (Edson) Fitzp. The Sequence Was Also Studied In Vitro, to identify the responsible Factors.Several amino acids, sugars or volatile compounds elicited one of more stages of the sequence, but only glutamic acid and aspartic acid elicited all stages and thus could account for the observed behaviour on roots. Polyuronates of root surface slime also may be needed for encystment and orientation of Spores, because germ‐tubes emerged towards roots from a fixed point near the former position of the water‐expulsion vacuole. However, identical patterns of zoospore accumulation and encystment on untreated roots and roots encased in alginate gel indicated that neither polyuronates nor Specific sugar residues of root slime caused localized encystment on roots.Based on these results, a ‘simplest case’ model of the pre‐penetration sequence on roots is proposed.

Direct detection of cell surface receptors for glycosyl residues on Phytophthora cinnamomi using polysaccharide-coated fluorescent latex beads

We have confirmed the presence of a fucose-specific binding protein on the surface of zoospores, cysts and germinated cysts of Phytophthora cinnamomi Rands using a direct binding assay with polysaccharide-coated fluorescent latex beads. The specificity for fucose was established using a range of polysaccharides and proteoglycans. Only maize root slime- and fucoidan-coated beads bound to the surface of zoospores, cysts and germinated cysts. Binding could be blocked with fucose or fucose-containing polysaccharides. In addition, maize root slime-coupled beads which were pre-incubated with the fucose-specific lectin from Ulex europeaus (UEA-1) did not bind to the cell surface of fungal propagules.

Behaviour of Phytophthora cinnamomi Zoospores on Roots of Four Avocado Cultivars

AbstractScanning electron microscopy was used to study the behaviour of Phytophthora cinnamomi zoospores on the roots of three tolerant avocado cultivars. Duke 7, G6 and Martin Grande, and a susceptible Edranol cultivar. Zoospores were attracted to the region of cell elongation and encysted on the roots of all cultivars studied. Adhesion of the zoospores appeared to be aided by root slime. Cysts usually produced one germ tube which penetrated the root directly, or formed an appressorium‐like swelling before penetration occurred. Extensive growth of germ tubes occurred where zoospores germinated some distance behind the region of elongation. Cysts germinating behind this region often formed branched germ tubes and more than one appressorium‐like swelling. There were no clear differences in the type of pre‐penetration structures, formed by zoospore cysts, on the roots of the different avocado cultivars.

Structural analysis of secreted slime from wheat and cowpea roots

The water-soluble, high M r components of slime secreted from the roots of wheat and cowpea are composed primarily of carbohydrate (95.5 and 97.5% w/w, respectively and some protein (5 and 3% w/w, respectively). For wheat, arabinose, xylose, glucose and galactose are the major neutral monosaccharides whereas for cowpea, arabinose, galactose and glucose predominate. Fucose is a minor constituent of both wheat and cowpea root slimes. Cowpea root slime contains significantly more uronic acids than that from wheat roots (11.5 vs 4% w/w). Methylation analysis suggests the presence of a range of polymers in the root slimes. In general, their composition appears to reflect that characteristically found for cell wall preparations from dicotyledons and graminaceous monocotyledons. In addition, arabinogalactan-proteins are components of both root slimes.

Biochemical markers of embryogenesis in tissue cultures of the maize inbred B73

Stable embryogenic, organogenic and undifferentiated cell lines of the maize ( Zea mays L.) inbred B73 were used to assess the value of using isozyme analyses and the composition of secreted polysaccharides to identify embryogenic cells. Esterase, glutamate dehydrogenase, alcohol dehydrogenase and β-glucosidase all possessed developmentally regulated isozymes but only esterase and glutamate dehydrogenase could be used to distinguish between embryogenic and shoot-forming cultures. Embryogenic callus and suspension cultures secreted a musilagenous polysaccharide whose production was stimulated by 2,4-dichlorophenoxyacetic acid (2,4-D). This polysaccharide was different from root slime and corn hull gum and may be related to the ‘cementing layer’ in maize kernels.

Determination of pea (Pisum sativum L.) root lectin using an enzyme-linked immunoassay

Root lectins are believed to participate in the recognition between Rhizobium and its leguminous host plant. Among other factors, testing this hypothesis is difficult because of the very low amounts in which root lectins are produced. A double-antibody-sandwich enzyme-linked immunoassay, was used to determine nanogram quantities of pea lectin in root slime and salt extracts of root cell-wall material when pea seedlings were 4 and 7 d old. In addition, a critical NO 3 (-) concentration (20 mM) which inhibited nodulation was found, and the lectin present in root slime and salt extracts of root cell walls of 4- and 7-d-old peas supplied with 20 mM NO 3 (-) was comparatively determined. With the enzyme-linked immunoassay, lectin quantities ranging between 20 and 100 nanograms could be determined. The assay is not affected by monomeric mannose and glucose (pealectin haptens). The slime of the 4-d-old roots contained more lectin than the slime of the 7-d-old roots. Salt-extractable, cell-wall-associated lectin accumulated in the older roots. Nitrate affected slime and cell-wall production, and the extractability of cell-wall material in both age groups. The presence of NO 3 (-) increased lectin in the slime, most notably in the younger roots; the relative amount of lectin in the slime was almost doubled. The cell-wall-associated, salt-extractable lectin decreased two- to threefold compared with the control group.

The synthesis and transport of some plant glycoproteins

Maize root slime is secreted by the outer root-cap cells. It is a complex of at least three polysaccharides, two of which are acidic and contain a high proportion of fucose. The polysaccharides are assembled and secreted by the endoplasmic reticulum and dictyosomes of the cells. It has been found that the synthesis of fucose-containing oligosaccharides takes place by an assembly of at least nine sugar residues on a polyprenyl diphosphate acceptor and this occurs at the endoplasmic reticulum. In addition, a glycoprotein carrying a large molecular mass carbohydrate portion containing fucose is synthesized mainly within the Golgi apparatus, although synthesis may be initiated in the endoplasmic reticulum. This glycoprotein could be an intermediate in the synthesis of slime polysaccharide, and the carbohydrate moiety of the glycoprotein may be assembled by transglycosylations from the polyprenyl diphosphate oligosaccharides.

Adhesion of fungal zoospores to root surfaces is mediated by carbohydrate determinants of the root slime

Adhesion of Phytophthora cinnamomi zoospores to Zea mays roots depends on the integrity of the carbohydrate component of the secreted root surface slime. Adhesion is abolished by periodate oxidation of the root surface, enzymic removal of terminal fucosyl residues of the slime and by interaction of the same residues with a fucose-binding lectin.

Pea lectins and the recognition of Rhizobium leguminosarum

Pisum sativum cv. Rondo seeds were found to contain two lectins, one of which seemed to be the mannosylated derivative of the other. Hapten-inhibited binding to human A erythrocytes and to Rhizobium leguminosarum by both lectins seemed to be identical; appropriate haptens are (α-linked) sugars with unsubstituted C-4- and C-6-hydroxyls in the same stereochemical position as with glucose or mannose. Pea root slime contained at least two glucose-binding proteins, one of which is a Rhizobium leguminosarum-agglutinating lectin with the same electrophoretic mobility as the glycosylated seed lectin. Both glucose-binding protein fractions were co-purified with a carbohydrate fraction of glucan nature. The role of pea lectins in the recognition of Rhizobium leguminosarum is discussed.

Intracellular Localization of GDP-Fucose

The intracellular site of synthesis of the fucose-rich polysaccharide slime secreted by corn roots was localized by monitoring the distribution of GDP-fucose:polysaccharide fucosyl transferase activity in subcellular fractions of corn roots. Root tip sections were chopped in the presence of 0.56 molar sucrose and 100 millimolar Tris (pH 7.0). After a brief centrifugation, the homogenate was applied to a Sepharose 4B column (1.5 x 30 cm). The turbid, particulate portion of the supernatant fraction eluted at the void volume. Ninety per cent of the enzyme activity was found in the pooled particulate fractions. The particulate fraction was purified on linear sucrose gradients. Gradient fractions were characterized by buoyant density, 280 nanometer absorbance, electron microscope observation, and distributions of NADH-cytochrome c oxidoreductase and fucosyl transferase activities.TWO PEAKS OF FUCOSYL TRANSFERASE ACTIVITY WERE OBSERVED: one in fractions rich in smooth endoplasmic reticulum, and another in fractions rich in dictyosome cisternae and dictyosome vesicles. The data suggest that the synthesis of corn root slime begins in the endoplasmic reticulum and is completed in the dictyosomes prior to secretion; however, the possibility that more than one fucosyl transferase exists in corn roots is discusses.

The structure and function of glycoproteins synthesized during slime-polysaccharide production by membranes of the root-cap cells of maize (Zea mays).

The synthesis of the maize root slime polysaccharides was investigated by using [1-3H]-fucose as a marker for slime production. Three fractions were separated by centrifugation in a CsCl density gradient. Two of these were glycoproteins and occurred within the membranes of the cells of the root tip; the third was the slime polysaccharides. Radioactive pulse-chase experiments showed that the glycoproteins were precursors of the slime polysaccharides, and the carbohydrate portion of the glycoproteins had a similar composition to that of the free slime. The linkage between the protein and the carbohydrate of one of the glycoproteins was shown to be a xylose-threonine bond. It is postulated that the slime polysaccharides are synthesized and transported on proteins within the membrane system of the root tip.

The amounts and rates of export of polysaccharides found within the membrane system of maize root cells.

1. Maize seedling roots were incubated in vivo with d-[U-(14)C]glucose for 2, 5, 10, 15, 30 and 45min. The total incorporation of radioactivity into polysaccharide components in isolated fractions was investigated, and the pattern of incorporation into different polysaccharide components within the rough endoplasmic reticulum, Golgi apparatus and exported material was analysed. 2. The membrane compartments reached a saturation value of radioactivity in polysaccharide components by 30min incubation. Radioactivity in exported polysaccharide continued to increase after that time. The latter was formed and maintained by a steady-state turnover of polysaccharide synthesis and transport from the membrane system. 3. If the only access of the slime polysaccharide to the cell surface is via dictyosome-derived vesicles, the amount of slime components in the Golgi apparatus would have to be displaced every 0.3min in order to maintain the observed rates of increase in slime. This is in contrast with a displacement time of about 2.5min that is necessary for polysaccharide components in the Golgi apparatus to produce the observed increase in cell-wall material. The activity of the membrane system in the production of maize root slime is 8 times as great as that of the membrane system involved in cell-wall synthesis. 4. If the amount of polysaccharide material in the Golgi apparatus is maintained only by inflow of polymeric material from the rough endoplasmic reticulum the total amount of slime components in the rough endoplasmic reticulum would have to be displaced every 7min to maintain a constant amount in the Golgi apparatus. If the endoplasmic reticulum contributed directly to the cell surface in the synthesis of cell-wall material, displacement times necessary to maintain the observed rate of polymer production would be very slow.

The sites of synthesis and transport of extracellular polysaccharides in the root tissues of maize.

1. Subcellular fractionation of maize roots resulted in the isolation of the following enriched fractions: cell wall, dictyosome, smooth-membrane and rough-microsomal fractions. In addition, extracellular polysaccharide of the root slime was isolated. 2. Maizeseedling roots were incubated in vivo with d-[U-(14)C]glucose, and the pattern of incorporation of radioactivity into the polysaccharides of each fraction was investigated. 3. The differentiation of maize-root cells with respect to the synthesis of specific extracellular polysaccharide directly relates to the polysaccharide synthesized and transported within the membrane system of the cell. A fucose-containing polysaccharide, characteristic only of root slime, was present only in the membrane system of the root-tip region of the root. Regions of typical secondary wall development within the root were characterized by an increased incorporation of radioactivity into xylose of polysaccharide within the membrane system. 4. The incorporation of radioactivity into glucan polymers in the membrane fractions was very low in all regions of the root. Since in regions of secondary wall development greater than 60% of all radioactive incorporation was into a glucan polymer, it can be inferred that this polymer, most probably cellulose, is not synthesized or transported within the compartments of the membrane system. It is suggested that synthesis of cellulose occurs at the surface of the plasmalemma. 5. Maize-root cells contained 40 times more rough endoplasmic reticulum than dictyosome membrane. The relative specific radioactivities of each fraction indicated that polysaccharide was concentrated in the region of the Golgi apparatus, which showed a 100% increase in specific radioactivity compared with the rough endoplasmic reticulum. The Golgi apparatus can thus be regarded as a localized focal point on the synthetic and transport system of polysaccharide by the intracellular membrane compartments.