Root mucilage from pea and its utilization by rhizosphere bacteria as a sole carbon source.

Plant roots secrete a complex polysaccharide mucilage that may provide a significant source of carbon for microbes that colonize the rhizosphere. High molecular weight mucilage was separated by high-pressure liquid chromatography gel filtration from low molecular weight components of pea root exudate. Purified pea root mucilage generally was similar in sugar and glycosidic linkage composition to mucilage from cowpea, wheat, rice, and maize, but appeared to contain an unusually high amount of material that was similar to arabinogalactan protein. Purified pea mucilage was used as the sole carbon source for growth of several pea rhizosphere bacteria, including Rhizobium leguminosarum 8401 and 4292, Burkholderia cepacia AMMD, and Pseudomonas fluorescens PRA25. These species grew on mucilage to cell densities of three- to 25-fold higher than controls with no added carbon source, with cell densities of 1 to 15% of those obtained on an equal weight of glucose. Micromolar concentrations of nod gene-inducing flavonoids specifically stimulated mucilage-dependent growth of R. leguminosarum 8401 to levels almost equaling the glucose controls. R. leguminosarum 8401 was able to hydrolyze p-nitrophenyl glycosides of various sugars and partially utilize a number of purified plant polysaccharides as sole carbon sources, indicating that R. leguminosarum 8401 can make an unexpected variety of carbohydrases, in accordance with its ability to extensively utilize pea root mucilage.

NMR studies of molecular structure in fruit cuticle polyesters.

The cuticle of higher plants functions primarily as a protective barrier for the leaves and fruits, controlling microbial attack as well as the diffusion of water and chemicals from the outside environment. Its major chemical constituents are waxes (for waterproofing) and cutin (a structural support polymer). However, the insolubility of cutin has hampered investigations of its covalent structure and domain architecture, which are viewed as essential for the design of crop protection strategies and the development of improved synthetic waterproofing materials. Recently developed strategies designed to meet these investigative challenges include partial depolymerization using enzymatic or chemical reagents and spectroscopic examination of the intact polyesters in a solvent-swelled form. The soluble oligomers from degradative treatments of lime fruit cutin are composed primarily of the expected 10,16-dihydroxyhexadecanoic and 16-hydroxy-10-oxo-hexadecanoic acids; low-temperature HF treatments also reveal sugar units that are covalently attached to the hydroxyfatty acids. Parallel investigations of solvent-swollen cutin using 2D NMR spectroscopy assisted by magic-angle spinning yield well-resolved spectra that permit detailed comparisons to be made among chemical moieties present in the intact biopolymer, the soluble degradation products, and the unreacted solid residue.

L-Altruronic acid formed by epimerization of D-galacturonic acid methyl esters during saponification of citrus pectin.

While searching for oligosaccharides containing rhamnose residues in the endopolygalacturonase (EPG) digest of saponified citrus pectin, we found several oligomers containing, in addition to galacturonic acid, a sugar previously unreported in pectin. The 1- and 2-D 1H NMR spectra of the oligosaccharides were consistent with the sugar being a uronic acid with its 2- and 3-hydroxyls being axial and 4-hydroxyl being equatorial. MALDI-TOF mass spectrometry indicated that the oligomers consisted solely of uronic acids. Reduction of the uronic acids in the oligosaccharides converted them to galactose and altrose. The altrose was found to be the L enantiomer by comparison of its trimethylsilyl (-)-2-butyl glycosides to those of authentic D-altrose and a racemic mixture. The sugar was not found in oligosaccharides prepared from EPG digestion of citrus pectin deesterified with pectin methylesterase rather than saponification. Thus, it appears that during saponification, a small proportion of the methylesterified galacturonic acid residues in pectins is epimerized at C-5 leading to formation of L-altruronic acid residues.

Use of scavenger beads to remove excess labeling reagents from capillary zone electrophoresis samples.

In many cases, samples for capillary zone electrophoresis (CZE) are derivatized with a chromophore or fluorophore to enhance their detectability. To ensure efficient labeling, a large excess of labeling agent is often used, which leads to the presence of a large peak for unreacted reagent. Here we report that excess reagent can be reacted with "scavenger beads" carrying an appropriate functional group to remove it from the sample solution. We present examples of removal of aminonaphthalene mono-, di-, and trisulfonic acid from mixtures in which they were used to label mono- or oligosaccharides by reductive amination. Aldehyde-containing scavenger beads were made by oxidizing Sephadex G-50 beads with sodium periodate. These were added to the labeling reaction mixtures after the reductive amination of the sugars was complete. Almost complete elimination of the peak from the labeling agent could be achieved.

A computer-controlled variable light attenuator for protection and autoranging of a laser-induced fluorescence detector for capillary zone electrophoresis.

Photodetectors have a limited range over which they can measure light intensities for any particular setting. The intensity of light reaching the detector can be kept within this range by using a liquid crystal variable light attenuator controlled by a computer that continuously checks the amount of light reaching the detector and adjusts the attenuation to an appropriate level. Using such a system we have constructed an intensified charge-coupled device (ICCD) camera-based detector with a dynamic range of over six orders of magnitude which is never exposed to damaging or saturating light levels.

Scarcity or complete lack of single rhamnose residues interspersed within the homogalacturonan regions of citrus pectin.

Commercial citrus pectin containing galacturonic acid and rhamnose in a ratio of approximately 40:1 was saponified and then exhaustively digested with endopolygalacturonase (EPG). The products were separated by ultrafiltration into low-molecular-weight (LMW) and high-molecular-weight (HMW) fractions. The LMW fraction accounted for 80% of the starting material, but for only 10% of the total rhamnose. The molar ratio of galacturonic acid to rhamnose of the LMW fraction was 236, suggesting that very few small Rha-containing oligomers were generated by the EPG digestion. No distinct Rha-containing oligomers were found by various chromatographic analyses of the LMW fraction. The HMW fraction, which only accounted for 10% by weight of the starting pectin, contained more than 85% of the rhamnose. The ratio of GalA to Rha in the HMW fraction was 1.7:1 and partial acid hydrolysis of this fraction produced a series of oligomers consisting of GalA-Rha repeating units, suggesting that it contained rhamnogalacturonan, which has a backbone composed of GalA-Rha disaccharide repeating units. The HMW fraction also contained large amounts of arabinose and galactose, which probably originated from side chains linked to some of the rhamnose residues. We propose that commercial citrus pectin is composed of two regions: the predominant region consists of chains of uninterrupted 1,4-linked alpha-D-GalA residues with between 60-70% of the residues methyl esterified; and the other region consists of rhamnogalacturonan with a backbone composed of GalA-Rha disaccharide repeating units and neutral sugar side chains.

Detection and differentiation of pectic enzyme activity in vitro and in vivo by capillary electrophoresis of products from fluorescent-labeled substrate.

A sensitive assay is described for the detection of pectate-depolymerizing enzymes using capillary electrophoresis of a fluorescent end-labeled pectate oligomer. The labeled oligomer is allowed to react with the enzyme either in vitro or in vivo, such as inside the intercellular spaces of a cotton cotyledon, and after an appropriate incubation time the products are analyzed by capillary electrophoresis. The site and mode of action of the pectate-depolymerizing activity can be inferred from the products. Both endo- and exopolygalacturonase activity, and lyase activity, were distinguished. Since only the fluorescent oligomer and products from its labeled reducing end are detected, there is no interference from other compounds; only pectic enzyme activity is detected. By this type of analysis we can show that there is considerable endo- and exo-polygalacturonase activity in the intercellular spaces of cotton cotyledons.

Separation of 8-aminonaphthalene-1,3,6-trisulfonate (ANTS)-labeled oligomers containing galacturonic acid by capillary electrophoresis: application to determining the substrate specificity of endopolygalacturonases.

Slight modifications of published procedures (Stefansson, M. and Novotny, M., Carbohydr. Res. 1994, 258 1-9) allow separation by relative size of 8-amino-naphthalene-1,3,6-trisulfonate (ANTS)-labeled neutral and acidic oligosaccharides by electrophoresis in uncoated capillaries. The separations are performed at pH 2.5 to suppress both any charge from carboxylic acid groups on the oligosaccharides and electroosmotic flow. Utility of the procedure is demonstrated by its application to characterization of substrate specificity of endopolygalacturonases. The results show that both a fungal and a bacterial endopolygalacturonase need four adjacent nonesterified galacturonic acid residues in a pectin to be able to act.

Solubilization and partial characterization of extensin fragments from cell walls of cotton suspension cultures. Evidence for a covalent cross-link between extensin and pectin.

Extensin, a major hydroxyproline (Hyp)-rich glycoprotein in walls of cultured cells of dicotyledonous plants, is very difficult to solubilize. To learn about the nature of the insolubilization, we have tested the ability of a variety of selective hydrolytic methods, and combinations of them, to liberate extensin or fragments of extensin from suspension-culture cell walls. After the complete deglycosylation of cotton (Gossypium hirsutum L.) walls, trypsinization solubilized 80% of the Hyp. The sequences of three abundant peptides were: (a) serine-Hyp-Hyp-Hyp-Hyp-Hyp-Hyp-serine-Hyp-Hyp-lysine, (b) serine-Hyp-Hyp-Hyp-Hyp-valine-lysine, and (c) serine-Hyp-Hyp-serine-alanine-Hyp-lysine. After a sequential treatment of walls with endopolygalacturonase, cellulase, -73 degrees C anhydrous hydrogen fluoride solvolysis, and ammonium bicarbonate extraction, only sugars indicative of rhamnogalacturonan I and protein remained insoluble. Trypsin treatment of this residue liberated 50% of the Hyp. A significant proportion of rhamnogalacturonan-associated sugars co-solubilized and co-purified along with the extensin fragments following the trypsinization. By sodium dodecyl sulfate gel electrophoresis and gel filtration, the glycopeptides fell into two classes. One class contained distinctly sized molecules with relative molecular weights in the range of 4,000 to 24,000. The other class did not enter the resolving gel and was hetero-disperse. After complete deglycosylation by a 0 degrees C anhydrous hydrogen fluoride treatment, the first class was little affected in its electrophoretic mobility, whereas the larger heterogeneous material mostly entered the separating gel. After further trypsinization of the deglycosylated peptides and analysis by capillary zone electrophoresis, the peptides in both size classes were shown to contain the sequences described above. From our observations we suggest that cotton extensin becomes insolubilized into cell walls in part by pectin-protein cross-links in addition to the protein-protein (or protein-phenolic-protein) cross-links that have been repeatedly suggested.

Continuous postcolumn detection of underivatized polysaccharides in high-performance liquid chromatography by reaction with permanganate.

We describe a continuous postcolumn reaction system for colorimetric detection of carbohydrates suitable for use with both preparative and analytical HPLC separations. A fraction of the effluent from the column is mixed via a T-junction with a 0.02% solution of potassium permanganate in 3 M sulfuric acid. The mixture then passes through a reaction coil heated to 100 degrees C, and its absorbance at 525 nm is continuously monitored. Bleaching of the permanganate is proportional to the sugar concentration. The major advantages of the detection system are its mass rather than molar sensitivity and insensitivity to changes in nonoxidizable buffer concentrations. As little as 0.1 micrograms of sugar can be detected. These features make the system suitable for detection, with high sensitivity, of polysaccharides using gradient elution from ion-exchange columns.

An Early Indicator of Resistance in Barley to Russian Wheat Aphid.

During early stages of infestation by Russian wheat aphids (Diuraphis noxia [Mordvilko]; RWAs), barley (Hordeum vulgare L.) leaf cells collapsed and showed autofluorescence in the mesophyll and bundle sheath adjacent to the RWA stylet sheath. The response was visually similar to the hypersensitive cell death response, typical of resistance to microbial pathogens. Resistant barley produced significantly more collapsed, autofluorescent cells (CAC) than did susceptible barley. RWA stylet entry sites and sheath paths also fluoresced, making them easy to observe in whole leaf sections. The number of CAC increased with the number of RWAs and with the number of days of feeding in resistant plants. The CAC could be observed 1 d following infestation, making this the most rapid plant response toward the RWAs known to date. The response may be useful in screening for resistant plants and may provide insight into resistance mechanisms in barley.

Determination of the pattern of methyl esterification in pectin. Distribution of contiguous nonesterified residues.

A method is described for determining the distribution of contiguous nonesterified galacturonic acid residues within pectins. First, the esterified galacturonic acids are converted to galactose by reduction with sodium borohydride, then the glycosidic linkages of the resulting galactose residues are cleaved selectively by liquid HF solvolysis. Separation and quantitation of the resulting galacturonic acid containing oligomers reveals the proportion of each stretch of contiguous nonesterified galacturonic acid residues in the original pectin. The distribution of nonesterified GalA in a pectin fraction obtained from cotton suspension culture cell walls with approximately 50% esterification appears to be far from random.

"SpectraGraph" and "SpectraSort": mass spectral display and interpretation software for the Macintosh.

Two computer programs entitled "SpectraGraph" and "SpectraSort" have been written for te Apple Macintosh. SpectraGraph allows graphical display, manipulation, storage, and printing of an input mass spectrum list that has been imported from a mass spectrometer or entered manually. SpectraGraph gives the user the ability to display, normalize, and multiply different mass ranges, annotate peaks, and perform various other operations on the spectral display. Also the mass spectrum and other graphics may be copied to and from other Macintosh application documents. SpectraSort has been developed to aid in the interpretation of mass spectra, particularly those of biopolymers, by calculating the mass differences between peaks in a mass spectrum. The user then has the option of matching the mass differences with masses of fragments or residues stored in several user-definable look-up tables.

Determination of the degree of methyl esterification of pectins in small samples by selective reduction of esterified galacturonic acid to galactose.

A procedure for the determination of the degree of methyl esterification of pectin in virtually any sample is described. Samples were dissolved or suspended in 1 M imidazole buffer, pH 7.0, cooled on ice, and reduced with sodium borohydride. Quantitative reduction of samples was accomplished after 1 h using at least 20 mg sodium borohydride/mg sample. The degree of methyl esterification was determined by either the increase in galactose content as determined by GLC of the sugar or by the change in galacturonic acid content by colorimetric uronic acid analyses. Sample requirements were at least as low as 100 micrograms per determination by GLC or 2 to 3 mg per determination by colorimetric uronic acid analysis compared to 5 mg or more per determination for other published procedures. The degrees of methyl esterification determined by the methods described have compared very favorably with those determined by established methods.

Separation and quantitation of galacturonic acid oligomers from 3 to over 25 residues in length by anion-exchange high-performance liquid chromatography.

A method for separation and quantitation of galacturonic acid oligomers from 3 to over 25 residues in length is described. Oligomers were labeled at the reducing end with 2-aminopyridine and then analyzed by anion-exchange high-performance liquid chromatography using a sodium acetate gradient. The amount of each oligogalacturonide present was determined by comparison to the response of an internal reference oligogalacturonide over a range from 0.5 to 20 nmol per oligomer. At least 5 h of incubation in the 2-aminopyridine reagent was required to obtain maximum and oligomer length-independent derivatization. To be analyzed using this technique, oligogalacturonides must possess a reducing terminus, they should be deesterified prior to derivatization if identification of the actual galacturonide chain length is desired, and they should fall within the range of 3 to over 25 galacturonide residues per oligomer. The wide range of oligogalacturonides separable, sensitivity of detection, ease of quantitation of chromatographic data, and ability to hydrolyze the 2-aminopyridinyl group from sugars makes this technique of potential use for numerous applications ranging from simple characterization of oligogalacturonide mixtures to purification of oligomers for use in bioassays.

Serum antibody response to carbohydrate antigens of Pasteurella haemolytica serotype 1: relation to experimentally induced bovine pneumonic pasteurellosis.

The antibody responses to the capsular carbohydrate (CC) purified from Pasteurella haemolytica serotype 1 were determined by an ELISA, using 135 sera from 6 calves vaccinated with phosphate-buffered saline solution, formalin-killed P haemolytica bacterins, live P haemolytica, or an extract of P haemolytica referred to as carbohydrate-protein subunit (CPS). Calves vaccinated with live P haemolytica, bacterins, or CPS developed serum antibodies to CC. Bacterins containing Freund incomplete adjuvant or Freund complete adjuvant induced higher antibody responses than did bacterins containing aluminum hydroxide. In 4 of 6 experiments, high antibody responses to CC were significantly (P less than 0.05) correlated with resistance to transthoracic challenge exposure with P haemolytica. When calves were challenge exposed with a dose of P haemolytica that was 4.5 times greater than the standard challenge exposure dose or when calves that had been vaccinated with CPS were challenge exposed, antibody responses did not significantly (P greater than 0.05) correlate with resistance to challenge exposure. The amount of serum antibodies to CPS increased significantly (P less than 0.05) when calves were vaccinated with live or killed P haemolytica or with CPS, compared with that in calves given saline solution. In 5 of 6 experiments, correlation between high antibody responses and resistance to challenge exposure was significant (P less than 0.05). The correlation between those variables was not significant (P less than 0.07) for CPS-vaccinated calves. In the ELISA, treatment of CPS with sodium m-periodate, to oxidize periodate-sensitive carbohydrate epitopes, failed to markedly alter the antibody response to CPS.(ABSTRACT TRUNCATED AT 250 WORDS)