Matrix-assisted ultraviolet laser-desorption ionization and electrospray-ionization time-of-flight mass spectrometry of sulfated neocarrabiose oligosaccharides

Several commercial sulfated neocarrabiose oligosaccharides were analyzed by matrix-assisted ultraviolet laser-desorption ionization time-of-flight mass spectrometry (UV-MALDI-TOF-MS). UV-MALDI-TOF-MS was carried out in the linear and reflectron modes and, as routine, in both the positive- and negative-ion modes. 2,5-Dihydroxybenzoic acid and nor-harmane were used as matrices. In the positive- and negative-ion modes, with both matrices, peaks corresponding to (M+Na)(+) and (M-Na)(-) ions, respectively, were obtained, with only some signals due to glycosidic linkage cleavages (prompt fragmentation). With 2,5-dihydroxybenzoic acid abundant matrix signals were observed; nor-harmane afforded very few matrix signals in both ion modes, but more desulfation (prompt fragmentation) of the compounds occurred. When the desorption/ionization process was highly efficient, the post-source decay (PSD) fragmentation patterns were also investigated; most of the fragments detected derived from glycosidic linkage cleavages. Electrospray-ionization time-of-flight mass spectrometry (ESI-TOF-MS) in the negative-ion mode confirmed, with the observation of the (M-Na)(-) and the multiply charged anions, the identity and the purity of the samples.     

Chromatography of permethylated oligosaccharide alditols

A new chromatographic method which enables the separation of permethylated oligosaccharide alditols has been developed. The method entails chromatography on precoated silica gel plates using benzene-methanol (16:1, v/v or 10:1 v/v) as developing solvent. Separations of disaccharides were obtained on the basis of glycosidic linkage and anomeric configuration; the method can accomodate oligosaccharides containing up to 15 glycose units. The combined use of thin-layer chromatography and gas-liquid chromatography provides a powerful approach for the characterization of oligosaccharides. Retention indices are given of permethylated oligosaccharide alditols on a fused-silica capillary column bonded with DB-1.     

Structural Analyses of Xyloglucan Heptasaccharide by the Post-source Decay Fragment Method Using Matrix-assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

In the post-source decay (PSD) fragment spectrum of a reduced xyloglucan heptasaccharide (XXXGol) from tamarind seeds, eleven sodium-adduct fragment ions and a precursor ion [M + Na]⁺ were clearly observed by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). Each fragment ion interval corresponded to the absence of unhydroxylose, unhydroglucose, and glucitol residues, indicating that PSD fragmentation cleavage in the sugar compound occurred only at glycosidic linkages close to the oxygen atom of saccharide ring members, and not in inner sugar ring bonds. The PSD fragment ions were classified into two series, one involving the reducing end and the other involving the non-reducing end. Structural information from both the reducing and non-reducing ends could therefore be simultaneously obtained from the measurement of the positive ion mode. Almost all the fragment ions from species larger than trisaccharide residues could be detected in this PSD fragment experiment. Such fragmentation information will enable the structural determination of xyloglucan oligosaccharides.     

Identification and structural analysis of synthetic oligosaccharides of Shigella sonnei using MALDI-TOF MS

MALDI-TOF mass spectroscopy was used for the molecular weight determination of protected synthetic oligosaccharides related to a cell surface bacterial polysaccharide. By-products containing chlorinated protecting groups caused isotopic patterns characteristic of the natural isotopic distribution of chlorine, were identified on the basis of isotopic distribution. 2,4,6-Trihydroxyacetophenone (THAP) as a matrix was better than 2,5-dihydroxybenzoic acid (DHB) for compounds containing chlorine, since monoisotopic resolution and no fragmentation were observed. In the post source decay (PSD) mode the identification of the oligosaccharide sequence through cleavage of the interglycosidic linkages was also possible, thus providing a sensitive and accurate tool for the structural verification of synthetic oligosaccharide intermediates.     

Matrix-assisted ultraviolet laser desorption/ionization time-of-flight (UV-MALDI-TOF) mass spectra of N-acylated and N,O-acylated glycosylamines

Matrix-assisted ultraviolet laser desorption/ionization time-of-flight mass spectrometry (UV-MALDI-TOF-MS) has shown to be a very useful technique for the study of the non-volatile and thermally non-stable N-acylated glycopyranosyl- and glycofuranosyl-amines. Of the several matrices tested, 2,5-dihydroxybenzoic acid (DHB) was the most effective giving good spectra in the positive-ion mode. In the linear and reflectron modes, the [M+Na](+) ions appeared with high intensity. Their fragmentation patterns were investigated by post-source decay (PSD) UV-MALDI-TOF-MS showing mainly cross-ring cleavages. In addition, N,O-acylated glycopyranosyl- and glycofuranosyl-amines were also analyzed by this technique. PSD UV-MALDI-TOF-MS gave significant signals for several primary fragment ions, which were proposed but not detected, or observed with very low abundance, in electron ionization mass spectrometry (EI-MS) experiments.     

Structural characterization of trace stilbene glycosides in Lysidice brevicalyx Wei using liquid chromatography/diode-array detection/electrospray ionization tandem mass spectrometry

The mass fragmentation patterns of stilbene glycosides isolated from the genus Lysidice were investigated by negative ion electrospray ionization tandem mass spectrometry, and the influence of collision energy on their fragmentation behavior is discussed. It is found that the presence of the Y₀⁻ and B₀⁻ ions in the MS² spectra is characteristic for 1 → 6 linked diglycosyl stilbenes, while the Y₀⁻, Y₁⁻, and Z₁⁻ ions are representative ions of 1 → 2 linked diglycosyl stilbenes. These results indicate that ESI-MSⁿ in the negative ion mode can be used to differentiate 1 → 6 and 1 → 2 linked diglycosyl stilbenes. Based on the fragmentation rules, 9 new trace constituents were identified or tentatively characterized in a fraction of Lysidice brevicalyx by using HPLC/HRMS and HPLC-DAD/ESI-MSⁿ. The results of the present study can assist in on-line structural identification of analogous constituents and targeted isolation of novel compounds from crude plant extracts.     

Structural analysis of the xyloglucan from Phaseolus coccineus cell-walls using cellulase-derived oligosaccharides

Oligosaccharides, obtained by digestion of a xyloglucan from the cell walls of Phaseolus coccineus with cellulase, have been isolated by gel filtration. Oligosaccharides containing 2–6 residues accounted for ～57% of the hydrolysate, with larger oligosaccharides (d.p. ～10) and partially degraded xyloglucan accounting for ～32% of the polymer. The major glycosidic linkages were determined by methylation analysis. Methylated penta- and hexa-saccharide alditols were isolated by reverse-phase h.p.l.c. and characterised by e.i.-m.s. and f.a.b.-m.s. Methylated derivatives of the di-, tri-, and tetra-saccharide alditols were examined by g.l.c.-m.s. in the e.i. and c.i. modes. A structure based on these results is proposed.     

Effects of elevated CO2 on the vasculature and phenolic secondary metabolism of Plantago maritima

We have examined the effect of elevated CO₂ on the vasculature and phenolic secondary metabolism on clones of the maritime plant Plantago maritima (L.). Plants were exposed to either ambient (360 μmol CO₂ mol⁻¹) or elevated (600 μmol CO₂ mol⁻¹) atmospheric CO₂ within a Solardome facility and harvested after 12 months' growth. Histochemical analysis of the leaves identified increases in the diameter of the minor leaf vein and associated lignified vessels in plants exposed to elevated CO₂. In the roots the number of lignified root vessels and stele width were also increased, but overall the lignified vessel-wall thickness was reduced in plants exposed to elevated CO₂, compared to those grown under ambient CO₂. To investigate whether or not these subtle changes in lignification were associated with perturbations in phenolic metabolism, aromatic natural products were analysed by HPLC-MS after treatment with cellulase to hydrolyse the respective glycosidic conjugates. The phenylpropanoids p-coumaric acid, caffeic acid, ferulic acid and the flavone luteolin were identified, together with the caffeoyl phenylethanoid glycosides, verbascoside and plantamajoside which were resistant to enzymatic digestion. Exposure to enhanced CO₂ resulted in subtle changes in the levels of individual metabolites. In the foliage a one-year exposure to enhanced CO₂ resulted in an increased accumulation of caffeic acid, whilst in the roots p-coumaric acid and verbascoside were enhanced. Our results suggest that significant changes in the vasculature of P. maritima on exposure to increased CO₂ are associated with only minor changes in the leaves of specific lignin-related metabolites.     

Rapid degradation of lindane (��-hexachlorocyclohexane) at low temperature by Sphingobium strains

Bioremediation of anthropogenic organic pollutants in cold climates is often limited by lower microbial or enzyme activity induced by low temperature. The present study addressed this issue through the degradation of ??-hexachlorocyclohexane (??-HCH) by three Sphingobium strains (S. indicum B90A, S. japonicum UT26 and S. francense Sp+) under low temperature (4 °C). After 5 days incubation at 4 °C, 79.7% and 43.8% of 5 and 25 mg L⁻¹ of ??-HCH added were degraded, respectively by the inoculation of 1.75 × 10⁷ cells mL⁻¹ of S. indicum B90A. An increase in inoculum concentration to 1.72 × 10⁸ cells mL⁻¹ significantly increased the degradation to 98.1 ± 1.7% of 5 mg L⁻¹ within 24 h. Further, S. indicum B90A and S. japonicum UT26 can rapidly degrade ??-HCH at 4 °C, while the degradation capability of S. francense Sp+ is relatively low. At 4 °C, ??-HCH is transformed to extremely low amounts of 1,2,4-trichlorobenzene (1,2,4-TCB) and 2,5-dichlorophenol (2,5-DCP) by S. indicum B90A, but most of ??-HCH were transformed to 2,5-Dichloro-2,5-cyclohexadiene-1,4-diol (2,5-DDOL) by S. japonicum UT26. These results revealed that haloalkane dehalogenases in some Sphingobium species are very active at temperature as low as 4 °C and S. indicum B90A might be a good candidate for developing novel bioremediation techniques for cold regions to decontaminate ??-HCH from soils/waters.     

A gas chromatographic method for the determination of aldose and uronic Acid constituents of plant cell wall polysaccharides

A major problem in determining the composition of plant cell wall polysaccharides has been the lack of a suitable method for accurately determining the amounts of galacturonic and glucuronic acids in such polymers. A gas chromatographic method for aldose analysis has been extended to include uronic acids. Cell wall polysaccharides are depolymerized by acid hydrolysis followed by treatment with a mixture of fungal polysaccharide-degrading enzymes. The aldoses and uronic acids released by this treatment are then reduced with NaBH₄ to alditols and aldonic acids, respectively. The aldonic acids are separated from the alditols with Dowex-1 (acetate form) ion exchange resin, which binds the aldonic acids. The alditols, which do not bind, are washed from the resin and then acetylated with acetic anhydride to form the alditol acetate derivatives. The aldonic acids are eluted from the resin with HCl. After the resin has been removed, the HCl solution of the aldonic acids is evaporated to dryness, converting the aldonic acids to aldonolactones. The aldonolactones are reduced with NaBH₄ to the corresponding alditols, dried and acetylated. The resulting alditol acetate mixtures produced from the aldoses and those from the uronic acids are analyzed separately by gas chromatography. This technique has been used to determine the changes in composition of Red Kidney bean (Phaseolus vulgaris) hypocotyl cell walls during growth, and to compare the cell wall polysaccharide compositions of several parts of bean plants. Galacturonic acid is found to be a major component of all the cell wall polysaccharides examined.     

Differentiation of isomeric Lewis blood groups by positive ion electrospray tandem mass spectrometry

Lewis histo-blood group antigens are one of the major classes of biologically active oligosaccharides. In this work, underivatized Lewis blood groups were studied by electrospray tandem mass spectrometry (ESI-MSⁿ) in the positive mode with three different mass analyzers: Q-TOF (quadrupole time-of-flight), QqQ (triple quadrupole), and LIT (linear ion trap). It was observed that, under collision-induced fragmentations, type 1 Lewis antigens (Le^a and Le^b) could be distinguished from type 2 (Le^x and Le^y) on the basis of specific fragmentations of the GlcNAc unit. Whereas O-4-linked sugars of the GlcNAc are lost as residues, the O-3-linked sugars undergo fragmentation both as sugar units and as sugar residues (unit −18 Da). Type 2 Lewis antigens also showed a characteristic cross-ring cleavage ^0,2A₂ of the GlcNAc. As a result, the product ions at m/z 388 and 305, characteristic of Le^x, and m/z 372, characteristic of Le^a, are proposed to distinguish the trisaccharide isomers Le^x/Le^a. Also, the product ions at m/z 534 and 305, characteristic of Le^y, and m/z 372, characteristic of Le^b, are proposed to distinguish the tetrasaccharide isomers Le^b/Le^y. These diagnostic fragment ions were further applied in the identification of Lewis type 2 antigens (Le^x and Le^y) in the lipopolysaccharide of the human gastric pathogen, Helicobacter pylori.     

Exploiting Natural Variation of Secondary Metabolism Identifies a Gene Controlling the Glycosylation Diversity of Dihydroxybenzoic Acids in Arabidopsis thaliana

Plant secondary metabolism is an active research area because of the unique and important roles the specialized metabolites have in the interaction of plants with their biotic and abiotic environment, the diversity and complexity of the compounds and their importance to human medicine. Thousands of natural accessions of Arabidopsis thaliana characterized with increasing genomic precision are available, providing new opportunities to explore the biochemical and genetic mechanisms affecting variation in secondary metabolism within this model species. In this study, we focused on four aromatic metabolites that were differentially accumulated among 96 Arabidopsis natural accessions as revealed by leaf metabolic profiling. Using UV, mass spectrometry, and NMR data, we identified these four compounds as different dihydroxybenzoic acid (DHBA) glycosides, namely 2,5-dihydroxybenzoic acid (gentisic acid) 5-O-β-D-glucoside, 2,3-dihydroxybenzoic acid 3-O-β-D-glucoside, 2,5-dihydroxybenzoic acid 5-O-β-D-xyloside, and 2,3-dihydroxybenzoic acid 3-O-β-D-xyloside. Quantitative trait locus (QTL) mapping using recombinant inbred lines generated from C24 and Col-0 revealed a major-effect QTL controlling the relative proportion of xylosides vs. glucosides. Association mapping identified markers linked to a gene encoding a UDP glycosyltransferase gene. Analysis of Transfer DNA (T-DNA) knockout lines verified that this gene is required for DHBA xylosylation in planta and recombinant protein was able to xylosylate DHBA in vitro. This study demonstrates that exploiting natural variation of secondary metabolism is a powerful approach for gene function discovery.     

The behavior of cellulose,amylose, and β-d-xylan towards anhydrous hydrogen fluoride

Cellulose, amylose, and d-glucose are converted into α-d-glucopyranosyl fluoride (3) when dissolved in anhydrous hydrogen fluoride. The fluoride subsequently undergoes condensation to afford a mixture of ligosaccharides, probably via an oxocarbonium ion. The fluoride 3 and the oligosaccharides are in an equilibrium, which was studied by ¹³C-n.m.r. spectroscopy; in dilute solution in hydrogen fluoride, the d-glucosyl fluoride is the main product present, but when the hydrogen fluoride is evaporated, the equilibrium is shifted towards the oligosaccharides. These constitute a complex mixture which was studied by methylation and subsequent analysis of the methylated alditols derived therefrom. (1→4)-β-d-Xylan and d-xylose behave similarly to the d-glucose derivatives towards hydrogen fluoride.     

A computational study of structure-reactivity relationships in Na-adduct oligosaccharides in collision-induced dissociation reactions

Elucidating the fragmentation mechanisms in oligosaccharides using theoretical calculations is useful in analyzing the experimentally obtained mass spectra. Semi-empirical and ab initio quantum mechanics calculations were used to study the relationship between the structure and reactivity and the chemical properties of oligosaccharides. In these calculations, sodium-cationized oligosaccharides were investigated to determine Na+ ion affinity at several binding positions; in addition, the dependence of the glycosidic bond cleavage on the Na+ position was examined. The calculated structures reported in this study are directed at interpreting experimentally observed fragment ions, resulting from the cleavage of the glycosidic bonds. The calculated results for oligosaccharides containing between three and five monosaccharide units (27 oligosaccharides) were compared with experimental data generated by matrix-assisted laser-desorption/ionization (MALDI) using a quadrupole ion trap (QIT) with a time-of-flight (TOF) mass spectrometer (MS).     

Seasonal dynamics of allelopathically significant phenolic compounds in globally successful invader Conyza canadensis L. plants and associated sandy soil

The seasonal dynamics of total phenolics and phenolic acids in the stems of the global invader Conyza canadensis, from March (young plants in the form of rosettes) to September (fruit abscission and the beginning of plant decline), and in sandy soil were monitored monthly in non-native areas. The highest amount of total free phenolics was found in its tissues (31,000 μg g⁻¹) during the flowering and fruiting time (August). Bound phenolics peaked (up to 8443 μg g⁻¹) during shoot elongation and intensive plant growth (May–June) and in September. In the stems, bound phenolic acids (p-coumaric, ferulic, p-hydroxybenzoic, vanillic and syringic) have a maximum twice, in May and in August, with ferulic acid predominating (up to 951.6 μg g⁻¹). Free phenolic acids in the plant's tissue peaked in May (plant elongation). In the soil under C. canadensis, the amount of bound phenolics decreased between March and June, before increasing up to the full bloom phase of the plants (August). The amount of bound phenolic acids was several times greater than that of free ones, with maximum values in August. C. canadensis is a highly important source of phenolics in the ruderal phytocoenosis in new areas. In order to better explain the mechanisms of the spread and domination of invasive plants in non-native areas, in which allelopathy plays a decisive role, it is necessary to measure the production of allelochemicals in tissue and their accumulation in soil at the shortest possible intervals and link this with the phases of plant development.     

Asexual propagation in the coral reef macroalga Halimeda (Chlorophyta, Bryopsidales): production, dispersal and attachment of small fragments

Siphonous, green macroalgae of the genus Halimeda are ubiquitous and ecologically important in tropical and subtropical marine environments. It has been hypothesized that the abundance of Halimeda on coral reefs is in part due to the ability of this genus to propagate asexually via vegetative fragmentation. However, vegetative fragmentation has only been documented for H. discoidea in a laboratory setting. To test the hypothesis that vegetative fragmentation contributes to field populations of Halimeda, we examined three aspects of fragmentation by H. tuna (Ellis and Solander) Lamouroux, H. opuntia (Linneaus) Lamouroux and H. goreaui Taylor on Conch Reef in the Florida Keys: (1) short-term (8 days) and long-term (14 weeks) fragment survival and rhizoid production in the laboratory and field (7 and 21 m), (2) size of the fragment pool and (3) influences of herbivory and water motion on production and dispersal of fragments. Although morphologically similar to H. discoidea, only a small percentage of H. tuna fragments survived. Fragments of H. opuntia and H. goreaui were more robust, and survival and rhizoid production were positively correlated with size in short-term trials. In 14-week field trials, one-third or fewer fragments of any species survived at 7 m, potentially because fragments were covered by large amounts of sediment. Survivors included some buried, seemingly dead individuals that turned green when exposed to light, highlighting the remarkable ability of this genus to survive disturbances. There was much less sediment accumulation at 21 m, where more fragments survived. Most (93%) eight-segment fragments of H. opuntia produced attachment rhizoids by the end of the 14-week trial. Overall, a range of 4.7-9.4 fragments of Halimeda m⁻² day⁻¹ were found on Conch Reef; most fragments were generated by H. goreaui. Fish bite marks were evident on 75-85% of the individuals of H. tuna and the number of bites per thallus ranged from 1 to 23. Herbivorous reef fish commonly fed on all three species of Halimeda. Some fish consumed the biomass, while others rejected most bites. For example, 83% of bites were rejected by the blue-striped grunt. Dispersal distances for rejected bites ranged from 0 to 31 m. Water motion was also responsible for fragment dispersal; experimentally produced fragments moved up to 48 cm day⁻¹. Results presented here suggest that asexual propagation of fragments of Halimeda is an important component of the life-history of this genus and vegetative fragmentation contributes to the abundance of this genus on coral reefs.     

Hydrolysis of low-molecular-weight oligosaccharides and oligosaccharide alditols by pig intestinal sucrase/isomaltase and glucosidase/maltase

The ability of purified pig intestinal sucrase/isomaltase (SI; EC 3.2.1.10/48) and glucosidase/maltase (GM; EC 3.2.1.20) to hydrolyze di- and oligosaccharides consisting of D-glucose and D-fructose residues and the corresponding alditols was studied. The products, after incubation, reflect different binding patterns at both catalytic sites of SI. The active site of the sucrase subunit cleaves alpha,beta-(1-->2) glycosidic bonds, and only two monomer units of the substrates bind with favorable affinity. Oligosaccharides and reduced oligosaccharides containing alpha-(1--6) and alpha-(1-->1) glycosidic bonds are hydrolyzed by isomaltase, and for the active site of this subunit more than two subsites were postulated. Moreover, different binding sites for various aglycons seem to exist for isomaltase. Oligosaccharide alcohols are cleaved at lower rates if the reduced sugar residue occupies the aglycon binding site. GM also hydrolyzes alpha-(1-->1) linkages, but at a lower rate. The enzyme has the ability to bind compounds containing residues other than D-glucose. There are indications for similarities between GM and the isomaltase subunit of SI in the binding mode of oligosaccharides.     

A novel mixed-mode solid phase extraction for simultaneous determination of melamine and cyanuric acid in food by hydrophilic interaction chromatography coupled to tandem mass chromatography

Utilizing a solid phase extraction column (MCT) containing mixed hydrophilic functional gel and cation exchange sorbent, a sensitive and rapid HPLC–MS/MS method for simultaneously determining the residues of melamine (MEL) and cyanuric acid (CYA) in human foodstuffs was developed. MEL and CYA in egg, pork, liver, kidney and pork, shrimp, sausage casing, honey, soybean milk, soybean powder and dairy product were extracted using acetonitrile/water, defatted with hexane and isolated using MCT solid phase extraction column. The residues were separated upon a hydrophilic interaction liquid chromatography (HILIC) column and analyzed by electrospray ionization under negative–positive switched mode on a triplequadrupole mass spectrometry. The selected reaction monitoring was performed on [M+H]⁺ of m/z 127.9 to provide the transition of 127 > 85 and 127 > 68 (MEL) while the [M−H]⁻ of m/z 127.1 was selected as the precursor ion for CYA resulting in product ions m/z 85 and 42. Isotope labeled internal standard (¹⁵N₃-MEL and ¹³C₃-CYA) and matrix-matched calibration were both used to observe the recovery to be 70.0–129.6% and 70.0–128.9% with RSD of 1.4–23.3% and 1.5–21.7% for MEL and CYA, respectively (n = 6). All the LODs and LOQs of MEL and CYA were less than 39.4 and 99.1 μg kg⁻¹, respectively, in 18 matrices, which were sensitive enough for quantitative analysis. This method has been proven effective in simultaneous determination of melamine and cyanuric acid when inspecting unknown and positive samples.     

β-Galactosidase from Lactobacillus plantarum WCFS1: biochemical characterization and formation of prebiotic galacto-oligosaccharides

Recombinant β-galactosidase from Lactobacillus plantarum WCFS1, homologously over-expressed in L. plantarum, was purified to apparent homogeneity using p-aminobenzyl 1-thio-β-d-galactopyranoside affinity chromatography and subsequently characterized. The enzyme is a heterodimer of the LacLM-family type, consisting of a small subunit of 35 kDa and a large subunit of 72 kDa. The optimum pH for hydrolysis of its preferred substrates o-nitrophenyl-β-d-galactopyranoside (oNPG) and lactose is 7.5 and 7.0, and optimum temperature for these reactions is 55 and 60 °C, respectively. The enzyme is most stable in the pH range of 6.5-8.0. The K_m, k_cat and k_cat/K_m values for oNPG and lactose are 0.9 mM, 92 s⁻¹, 130 mM⁻¹ s⁻¹ and 29 mM, 98 s⁻¹, 3.3 mM⁻¹ s⁻¹, respectively. The L. plantarum β-galactosidase possesses a high transgalactosylation activity and was used for the synthesis of prebiotic galacto-oligosaccharides (GOS). The resulting GOS mixture was analyzed in detail, and major components were identified by using high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) as well as capillary electrophoresis. The maximal GOS yield was 41% (w/w) of total sugars at 85% lactose conversion (600 mM initial lactose concentration). The enzyme showed a strong preference for the formation of β-(1→6) linkages in its transgalactosylation mode, while β-(1→3)-linked products were formed to a lesser extent, comprising ∼80% and 9%, respectively, of the newly formed glycosidic linkages in the oligosaccharide mixture at maximum GOS formation. The main individual products formed were β-d-Galp-(1→6)-d-Lac, accounting for 34% of total GOS, and β-d-Galp-(1→6)-d-Glc, making up 29% of total GOS.     

Effect of Sodium Chloride on Fruit Ripening of the Nonripening Tomato Mutants nor and rin

Tomato (Lycopersicon esculentum Mill) plants of the nonripening mutant nor, the ripening-inhibited mutant rin, and the normal cultivar `Rutgers' were grown in nutrient solution supplemented with 3 grams per liter NaCl from the time of anthesis. In plants treated with NaCl, all the ripening parameters of the fruits of the nor mutant increased, but those of the rin mutant did not. The ripening of the fruits of the NaCl-treated nor plants was characterized by the development of a red color and taste, increased pectolytic activity, and increased evolution of CO₂ and ethylene. These changes do not normally take place in nor under control conditions. The values of these ripening parameters in nor were lower than those of the normal Rutgers fruits. In addition, both in nor and rin and in the normal variety, exposure of the plants to NaCl shortened the developmental period of the fruit, decreased the fruit size, and increased the concentrations of total soluble solids, Na⁺, Cl⁻, reducing sugars, and titratable acids in the fruit. The role of NaCl in overcoming the inability of nor to ripen is discussed.