Nasturtium Seeds Research Articles

Farklı Parametreler Kullanılarak Bazı Yenilebilir Çiçeklere Ait Tohumların Çıkış Oranlarının Değerlendirilmesi

In the study, the emergence power of 4 different edible flower seeds was determined using different parameters. It has been observed that final emergence percentage alone is not sufficient to express the emergence characteristics of the seed. When evaluated in terms of final emergence percentage, while calendula, red nasturtium and yellow nasturtium seeds had similar seed emergence, electric flower seeds had less emergence compared to the others. Considering the average emergence time, it was determined that nasturtium seeds emerged faster and more homogeneously compared to calendula and electric flower. In the study, germination index, emergence rate coefficient, emergence rate index values were calculated and it was determined that red and yellow nasturtium had better seed emergence in terms of these values compared to calendula and electric flower. As a result of the study, it was determined that final germination or emergence percentage alone was not sufficient to define the germination characteristics of seeds, and that determining at least one of the parameters such as germination index, emergence rate coefficient or emergence rate index in addition to the final emergence percentage would provide more reliable results.

Chemical Composition and Biological Activity of Essential Oil and Extract from the Seeds of Tropaeolum majus L. var. altum.

Plant Tropaeolum majus L. (garden nasturtium) belongs to the family Tropaeolaceae and contains benzyl glucosinolate. The breakdown product of benzyl glucosinolate, benzyl isothiocyanate (BITC), exhibits various biological activities such as antiproliferative, antibacterial and antiinflammatory. In order to optimize the content of biologically active volatile compounds in plant extract and essential oil, the use of appropriate extraction technique has a crucial role. The current study investigates the effect of two modern extraction methods, microwave-assisted distillation (MAD) and microwave hydrodiffusion and gravity (MHG), on the chemical composition of volatile components present in the essential oil and extract of garden nasturtium (T. majus L. var. altum) seeds. Investigation of the biological activity of samples (essential oil, extract and pure compounds) was focused on the antiproliferative effect against different cancer cell lines: cervical cancer cell line (HeLa), human colon cancer cell line (HCT116) and human osteosarcoma cell line (U2OS), and the antibacterial activity which was evaluated against the growth and adhesion of Staphylococcus aureus and Escherichia coli to polystyrene surface. Essential oil and extract of garden nasturtium (T. majus) seeds were isolated by two extraction techniques: MAD and MHG. BITC and benzyl cyanide (BCN) present in the extract were identified by gas chromatography-mass spectrometry. Essential oil of T. majus showed higher antiproliferative activity (IC50<5 µg/mL) than T. majus extract (IC50<27 µg/mL) against three cancer cell lines: HeLa, HCT116 and U2OS. BITC showed much higher inhibitory effect on all tested cells than BCN. The essential oil and extract of T. majus showed strong antimicrobial activity against S. aureus and E. coli. This work represents the first comparative report on the antiproliferative activity of the essential oil and extract of T. majus seeds, BITC and BCN against HeLa, HCT116 and U2OS cells as well as their antimicrobial activity against S. aureus and E. coli. This study demonstrates that the essential oil of T. majus seeds exhibits stronger antiproliferative and antimicrobial activity than the plant extract.

CHERNYAKHIV «BULLAS» (METAL AND LEATHER PENDANTS-FOLDING IN THE CHERNYAKHIV—SINTANA-DE-MURES CULTURE’S AREA)

Decorations of the Chernyakhiv—Sintana-de-Mures Culture (ChSM) includes metal and leather pendants which by some morphological features can be considered as derivatives of the roman bulla. Bulla is a small capsule pendant folded from two identical halves. It’s assumed that bulla originated with the Etruscans in the VIIth century BC. The bulla was later borrowed by romans, who originally intended it for boys — the children of aristocrats from the ninth day of birth to 14—16 years, until receiving the status of a Roman citizen. Later the golden bulla lost the meaning of a social marker and became mainly a property feature. Content of bulla were parts of plants, animal bones, pieces of textile, sand etc.&#x0D; Derivatives of the Roman bulla in the ChSM Culture have been found in next burial complexes (Barcea 139, Bila 3, Cherneliv-Rus’kyi 96, Dumaniv 8, Sosnova 245), leather bullas were found once (Legedzine 16). Besides, two pendants-capsules were found on late Scythians Lower Dnieper cemetery Mykolaivka (burial 64, grave 1), which contained ChSM Culture’s graves too.&#x0D; Following observation deserves the attention. The capacity of one pendant from Pannonia is similar by decor to clay knots, which were spread in many cultures, including ChSM Culture. They are interpreted as magic, used for fortune-telling and similar to nasturtium seeds.&#x0D; Contents of Chernyakhiv Culture’s «bullas» are unknown, but combination with another pendants-amulets certainly confirms the magical character of such pendants.&#x0D; Finally, metal bullae, cylindrical and flat cylindrical pendants-capsules continued their existence in the early Middle Ages and as metal folding icons and leather incenses survived in orthodox church to this day.

Influence of irrigation conditions in the germination of plasma treated Nasturtium seeds

Plasma treatments had emerged as a useful technique to improve seed germination. In this work we investigate the influence of different irrigation conditions and plasma treatments on the germination of nasturtium seeds. During plasma treatment, seeds experience a progressive weight loss as a function of treatment time that has been associated to water release, a process that is more pronounced after longer plasma treatment times. Seeds treated for short times (<30 s) are able to germinate more efficiently than untreated specimen under hydric stress (drought conditions), while plasma treatments for longer times (up to 300 s) impaired germination independently on irrigation conditions. Characterization analysis of plasma treated seeds by FTIR-ATR, SEM/EDX and XPS showed that plasma treatment affected the chemical state of pericarp while, simultaneously, induced a considerable increase in the seeds water uptake capacity. The decrease in germination efficiency found after plasma treatment for long times, or for short times under optimum irrigation conditions, has been attributed to that the excess of water accumulated in the pericarp hampers the diffusion up to the embryo of other agents like oxygen which are deemed essential for germination.

RNA-Seq Analysis of Developing Nasturtium Seeds (Tropaeolum majus): Identification and Characterization of an Additional Galactosyltransferase Involved in Xyloglucan Biosynthesis

A deep-sequencing approach was pursued utilizing 454 and Illumina sequencing methods to discover new genes involved in xyloglucan biosynthesis. cDNA sequences were generated from developing nasturtium (Tropaeolum majus) seeds, which produce large amounts of non-fucosylated xyloglucan as a seed storage polymer. In addition to known xyloglucan biosynthetic genes, a previously uncharacterized putative xyloglucan galactosyltransferase was identified. Analysis of an Arabidopsis thaliana mutant line defective in the corresponding ortholog (AT5G62220) revealed that this gene shows no redundancy with the previously characterized xyloglucan galactosyltransferase, MUR3, but is required for galactosyl-substitution of xyloglucan at a different position. The gene was termed XLT2 for Xyloglucan L-side chain galactosylTransferase position 2. It represents an enzyme in the same subclade of glycosyltransferase family 47 as MUR3. A double mutant defective in both MUR3 (mur3.1) and XLT2 led to an Arabidopsis plant with xyloglucan that consists essentially of only xylosylated glucosyl units, with no further substitutions.

Incorporation of β-(1,6)-linked glucooligosaccharides (pustulooligosaccharides) into plant cell wall structures

AbstractProtein extract of germinating nasturtium (Tropaeolum majus) seeds containing xyloglucan endotransglycosylase (xyloglucan xyloglucosyl transferase, EC 2.4.1.207, abbreviated XET) exhibited the heterotransglycosylating activity with donor/acceptor substrate pair xyloglucan/sulphorhodamine labelled pustulooligosaccharides (XG/PUOS-SR) in a dot blot assay. The heterotransglycosylating activity was confirmed by the substrate-product changes during transglycosylation by HPLC size-exclusion chromatography. Another donor substrate capable of being coupled with PUOS-SR was cellulose, probably owing to its structural similarity to xyloglucan. Surprisingly, microscopic comparison of the incorporation of the labelled xyloglucan nonasaccharide XGO9-SR (specific substrate for XET) and PUOS-SR into the cell wall structures clearly showed differences in their binding to specific cell structures: the primary cell wall and the plasma membrane. These findings indicate the existence in nasturtium of XETs with different localisation, substrate specificity and, probably, function.

Effect of the label of oligosaccharide acceptors on the kinetic parameters of nasturtium seed xyloglucan endotransglycosylase (XET)

Fluorescently labeled derivatives of a xyloglucan (XG) nonasaccharide Glc 4Xyl 3Gal 2 (XLLG) were used as glycosyl acceptors in assays of xyloglucan endotransglycosylase (XET) from germinated nasturtium ( Tropaeolum majus) seeds. We have investigated how the type of the oligosaccharide label influences the kinetic parameters of the reaction. The fluorescent probes used to label XLLG were anthranilic acid (AA), 8-aminonaphtalene-1,3,6-trisulfonic acid (ANTS), fluorescein isothiocyanate (FITC), and sulforhodamine (SR), respectively. The obtained data were compared with those of the reactions where aldose and/or alditol forms of tritium-labeled xyloglucan-derived nonasaccharide served as the respective acceptors. Modification at C-1 of the reducing-end glucose in XLLG by substitution with the fluorophore markedly affected the kinetic parameters of the reaction. The Michaelis constants K m for individual acceptors increased in the order [1- 3H]XLLG < XLLG-SR < [1- 3H]XLLGol < XLLG-FITC < XLLG-ANTS < XLLG-AA, while the turnover numbers characterized by k cat decreased in the order XLLG-FITC > XLLG-SR > XLLG-ANTS > [1- 3H]XLLGol > [1- 3H]XLLG > XLLG-AA. Catalytic efficiency (expressed as k cat/ K m) with XLLG labeled with SR or FITC was 15 and 28 times, respectively, higher than with the tritium-labeled natural substrate [1- 3H]XLLG. Comparison of the kinetic parameters found with acceptors labeled with different types of labels enables to select the most effective substrates for the high-throughput assays of XET.

Xyloglucan endotransglycosylases (XETs) from germinating nasturtium (Tropaeolum majus) seeds: Isolation and characterization of the major form

Five forms of xyloglucan endotransglycosylase/hydrolase (XTH) differing in their isoelectric points (pI) were detected in crude extracts from germinating nasturtium seeds. Without further fractionation, all five forms behaved as typical endotransglycosylases since they exhibited only transglycosylating (XET) activity and no xyloglucan-hydrolysing (XEH) activity. They all were glycoproteins with identical molecular mass, and deglycosylation led to a decrease in molecular mass from approximately 29 to 26.5 kDa. The major enzyme form having pI 6.3, temporarily designated as TmXET(6.3), was isolated and characterized. Molecular and biochemical properties of TmXET(6.3) confirmed its distinction from the XTHs described previously from nasturtium. The enzyme exhibited broad substrate specificity by transferring xyloglucan or hydroxyethylcellulose fragments not only to oligoxyloglucosides and cello-oligosaccharides but also to oligosaccharides derived from beta-(1,4)-d-glucuronoxylan, beta-(1,6)-d-glucan, mixed-linkage beta-(1,3; 1,4)-d-glucan and at a relatively low rate also to beta-(1,3)-gluco-oligosaccharides. The transglycosylating activity with xyloglucan as donor and cello-oligosaccharides as acceptors represented 4.6%, with laminarioligosaccharides 0.23%, with mixed-linkage beta-(1,3; 1,4)-d-gluco-oligosaccharides 2.06%, with beta-(1,4)-d-glucuronoxylo-oligosaccharides 0.31% and with beta-(1,6)-d-gluco-oligosaccharides 0.69% of that determined with xyloglucan oligosaccharides as acceptors. Based on the sequence homology of tryptic fragments with the sequences of known XTHs, the TmXET(6.3) was classified into group II of the XTH phylogeny of glycoside hydrolase family GH16.

A real-time fluorogenic assay for the visualization of glycoside hydrolase activity in planta.

There currently exists a diverse array of molecular probes for the in situ localization of polysaccharides, nucleic acids, and proteins in plant cells, including reporter enzyme strategies (e.g. protein-glucuronidase fusions). In contrast, however, there is a paucity of methods for the direct analysis of endogenous glycoside hydrolases and transglycosidases responsible for cell wall remodeling. To exemplify the potential of fluorogenic resorufin glycosides to address this issue, a resorufin beta-glycoside of a xylogluco-oligosaccharide (XXXG-beta-Res) was synthesized as a specific substrate for in planta analysis of XEH activity. The resorufin aglycone is particularly distinguished for high sensitivity in muro assays due to a low pK(a) (5.8) and large extinction coefficient (epsilon 62,000 M(-1) cm(-1)), long-wavelength fluorescence (excitation 571 nm/emission 585 nm), and high quantum yield (0.74) of the corresponding anion. In vitro analyses demonstrated that XXXG-beta-Res is hydrolyzed by the archetypal plant XEH, nasturtium (Tropaeolum majus) NXG1, with classical Michaelis-Menten substrate saturation kinetics and a linear dependence on both enzyme concentration and incubation time. Further, XEH activity could be visualized in real time by observing the localized increase in fluorescence in germinating nasturtium seeds and Arabidopsis (Arabidopsis thaliana) inflorescent stems by confocal microscopy. Importantly, this new in situ XEH assay provides an essential complement to the in situ xyloglucan endotransglycosylase assay, thus allowing delineation of the disparate activities encoded by xyloglucan endotransglycosylase/hydrolase genes directly in plant tissues. The observation that XXXG-beta-Res is also hydrolyzed by diverse microbial XEHs indicates that this substrate, and resorufin glycosides in general, may find broad applicability for the analysis of wall restructuring by polysaccharide hydrolases during morphogenesis and plant-microbe interactions.

Active-site Mapping of a Populus Xyloglucan endo-Transglycosylase with a Library of Xylogluco-oligosaccharides

Restructuring the network of xyloglucan (XG) and cellulose during plant cell wall morphogenesis involves the action of xyloglucan endo-transglycosylases (XETs). They cleave the XG chains and transfer the enzyme-bound XG fragment to another XG molecule, thus allowing transient loosening of the cell wall and also incorporation of nascent XG during expansion. The substrate specificity of a XET from Populus (PttXET16-34) has been analyzed by mapping the enzyme binding site with a library of xylogluco-oligosaccharides as donor substrates using a labeled heptasaccharide as acceptor. The extended binding cleft of the enzyme is composed of four negative and three positive subsites (with the catalytic residues between subsites -1 and +1). Donor binding is dominated by the higher affinity of the XXXG moiety (G=Glcbeta(1-->4) and X=Xylalpha(1-->6)Glcbeta(1-->4)) of the substrate for positive subsites, whereas negative subsites have a more relaxed specificity, able to bind (and transfer to the acceptor) a cello-oligosaccharyl moiety of hybrid substrates such as GGGGXXXG. Subsite mapping with k(cat)/K(m) values for the donor substrates showed that a GG-unit on negative and -XXG on positive subsites are the minimal requirements for activity. Subsites -2 and -3 (for backbone Glc residues) and +2' (for Xyl substitution at Glc in subsite +2) have the largest contribution to transition state stabilization. GalGXXXGXXXG (Gal=Galbeta(1-->4)) is the best donor substrate with a "blocked" nonreducing end that prevents polymerization reactions and yields a single transglycosylation product. Its kinetics have unambiguously established that the enzyme operates by a ping-pong mechanism with competitive inhibition by the acceptor.

Pectic homogalacturonan masks abundant sets of xyloglucan epitopes in plant cell walls

BackgroundMolecular probes are required to detect cell wall polymers in-situ to aid understanding of their cell biology and several studies have shown that cell wall epitopes have restricted occurrences across sections of plant organs indicating that cell wall structure is highly developmentally regulated. Xyloglucan is the major hemicellulose or cross-linking glycan of the primary cell walls of dicotyledons although little is known of its occurrence or functions in relation to cell development and cell wall microstructure.ResultsUsing a neoglycoprotein approach, in which a XXXG heptasaccharide of tamarind seed xyloglucan was coupled to BSA to produce an immunogen, we have generated a rat monoclonal antibody (designated LM15) to the XXXG structural motif of xyloglucans. The specificity of LM15 has been confirmed by the analysis of LM15 binding using glycan microarrays and oligosaccharide hapten inhibition of binding studies. The use of LM15 for the analysis of xyloglucan in the cell walls of tamarind and nasturtium seeds, in which xyloglucan occurs as a storage polysaccharide, indicated that the LM15 xyloglucan epitope occurs throughout the thickened cell walls of the tamarind seed and in the outer regions, adjacent to middle lamellae, of the thickened cell walls of the nasturtium seed. Immunofluorescence analysis of LM15 binding to sections of tobacco and pea stem internodes indicated that the xyloglucan epitope was restricted to a few cell types in these organs. Enzymatic removal of pectic homogalacturonan from equivalent sections resulted in the abundant detection of distinct patterns of the LM15 xyloglucan epitope across these organs and a diversity of occurrences in relation to the cell wall microstructure of a range of cell types.ConclusionThese observations support ideas that xyloglucan is associated with pectin in plant cell walls. They also indicate that documented patterns of cell wall epitopes in relation to cell development and cell differentiation may need to be re-considered in relation to the potential masking of cell wall epitopes by other cell wall components.

Molecular characterisation of a xyloglucan oligosaccharide-acting α-D-xylosidase from nasturtium (Tropaeolum majus L.) cotyledons that resembles plant 'apoplastic' α-D-glucosidases

We report the isolation, sequencing and analysis of the cDNA corresponding to an alpha-D-xylosidase involved in the mobilisation of xyloglucan from the cotyledons of germinated nasturtium (Tropaeolum majus L.) seeds. The translated open reading frame (2,808 bp including the stop codon), gave a polypeptide of 935 amino acids. It included the sequences of eleven peptides obtained by endo-proteinase digestion of the protein, and a putative hydrophobic signal sequence characteristic of a protein that is directed through the plasma membrane. The deduced molecular weight of the translated protein was appreciably higher than the molecular weight determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, suggesting post-translational modification. The protein sequence showed high homology (76.0% identity over 896 amino acids) with a putative alpha-xylosidase sequence from Arabidopsis thaliana and there was homology with several alpha-glucosidases, notably those associated with the plant cell apoplast. The enzyme is a member of Family 31 of the glycosyl hydrolases and it fits into Clade 1 of the phylogenic analysis of alpha-glucosidases. Although in vivo the nasturtium enzyme catalyses mobilisation of cell wall xyloglucan, the homology of its primary sequence with alpha-glucosidases prompted study of its action on a range of alpha-glucosides. It was active against several alpha-(1-->4)-and alpha-(1-->6)-linked substrates, the former being hydrolysed faster. The functional and evolutionary relationships between this alpha-D-xylosidase and plant "apoplastic" alpha-D-glucosidases are discussed.

Application of Fourier Transform Infrared Spectroscopy in Quantitation of Trierucin in High-Erucic-Acid Rapeseed Oils

Development of varieties of high-erucic-acid (HEAR) rapeseed with high levels of erucic acid (C22:1) would be valuable for use in oleochemical feedstocks. As an aid to this breeding effort, a rapid method to detect trierucin (C22:1 triacylglycerol), an indicator that erucic acid is being incorporated in all three positions of the triacylglycerol, would be useful. Fingerprint (1850–600 cm−1) Fourier transform infrared (FT-IR) spectra of HEAR oil extracted from Brassica napus L. cv. Mercury, trierucin, and varying amounts of trierucin spiked in this oil were recorded. The regions of 1790–1690 cm−1and 1485–990 cm−1were subjected to a partial least-squares (PLS) procedure to obtain a calibration equation [ R2= 0.992 and root-mean-squared deviation (RMSD) = 2.78]. The established calibration was then tested on the spectra of mixtures of another HEAR oil from B. napus cv. Turret and trierucin. The predicted values differ from the actual values with an RMSD of 1.6%. The calibration was also tested on high-erucic-acid oils from crambe ( Crambe abyssinica) and nasturtium ( Tropaeolum majus L.) seeds. For crambe, the value found (0.4%) was close to that reported in the literature, while the value of 75% for nasturtium oil was close to the value for total triacylglycerols with erucic acid in the 2-position as reported in the literature. FT-IR spectra of triolein (C18: 1 triacylglycerol), trieicosenoin (C20:1 triacylglycerol), and trinervonin (C24:1 triacylglycerol) compared with the FT-IR spectrum of trierucin revealed the uniqueness of IR spectrum of trierucin that forms the basis of PLS analysis of trierucin in HEAR oils.

Substrate subsite recognition of the xyloglucan endo-transglycosylase or xyloglucan-specific endo-(1-->4)-beta-D-glucanase from the cotyledons of germinated nasturtium (Tropaeolum majus L.) seeds.

We have investigated the substrate subsite recognition requirement of the xyloglucan endo-transglycosylase/xyloglucan-specific endo-(1-->4)-beta-D-glucanase (NXET) from the cotyledons of nasturtium seedlings. Seed xyloglucans are composed almost entirely of the Glc4 subunits XXXG, XLXG, XXLG and XLLG, where G represents an unsubstituted glucose residue, X a xylose-substituted glucose residue and L a galactosyl-xylose-substituted glucose residue. Thus in the xyloglucan sequence shown below, the xylose (Xyl) residues at the backbone glucose (Glc) residues numbered -3, -2, +2 and +3 may be galactose-substituted, and NXET cleaves between the unsubstituted glucose at -1 and the xylose-substituted glucose at +1, which never carries a galactosyl substituent. [formula: see text] We have isolated the xyloglucan oligosaccharides XXXGXXXG and XLLGXLLG from NXET digests of tamarind seed xyloglucan, have modified them enzymatically using a pure xyloglucan oligosaccharide-specific alpha-xylosidase from nasturtium seeds to give GXXGXXXG and GLLGXLLG, and have identified and compared the products of NXET action on XXXGXXXG, GXXGXXXG, XLLGXLLG and GLLGXLLG. We have also compared the molar proportions of XXXG, XLXG, XXLG and XLLG in native tamarind and nasturtium seed xyloglucans with those in NXET digests of these polysaccharides. Using these and existing data we have demonstrated that NXET action does not require xylose-substitution at glucose residues -4, -2, +1 and +3 and that xylose substitution at +2, is a requirement. There may also be a requirement for xylose substitution at -3. We have demonstrated also that galactosyl substitution of a xylose residue at +1 prevents, and at -2 modifies, chain-cleavage. A partial model for the minimum substrate binding requirement of NXET is proposed.

Molecular characterization of a xyloglucan-specific endo-(14)-beta-d-glucanase (xyloglucan endo-transglycosylase) from nasturtium seeds

Molecular characterization of a xyloglucan-specific endo-(14)-beta-d-glucanase (xyloglucan endo-transglycosylase) from nasturtium seeds

Molecular characterization of a xyloglucan-specific endo-(14)-beta-d-glucanase (xyloglucan endo-transglycosylase) from nasturtium seeds

A novel xyloglucan-specific endo-(1-->4)-beta-D-glucanase, involved in the post-germinative mobilization of xyloglucan storage reserves, has previously been isolated from nasturtium (Tropaeolum majus L.) seed. Its mode of action has been shown, in vitro, to be one of transglycosylation except at low substrate (glycosylacceptor) concentrations when hydrolysis predominates. Here it is shown that this nasturtium seed xyloglucan endo-transglycosylase is encoded by a single gene which is transcribed and processed to a 1.5 kb mRNA. The isolation and DNA sequence analysis of a cDNA copy of the nasturtium xyloglucan endo-transglycosylase transcript is described. The cDNA encodes a 33.5 kDa precursor polypeptide which is subsequently processed to a 31 kDa mature protein. The precursor incorporates an N-terminal signal sequence which probably contains information relevant to the targeting of the enzyme to the cell wall. The computer-predicted isoelectric point (5.14) and low (approximately 0%) alpha-helix content of the deduced mature protein are in excellent agreement with the experimental data obtained using the purified enzyme. The deduced protein sequence lacks homology with known plant endo-(1-->4)-beta-D-glucanases, consistent with the unique properties of the enzyme. Database searches have revealed that a Brassica protein (meri-5) of previously unknown function, but abundantly expressed in expanding tissue, shares structural identity with the nasturtium xyloglucan endo-transglycosylase. The expression of a xyloglucan endo-transglycosylase in expanding tissue would be consistent with the contention that enzymes of this type are involved in cell wall loosening.

Action of a pure xyloglucan endo-transglycosylase (formerly called xyloglucan-specific endo-(14)-beta-d-glucanase) from the cotyledons of germinated nasturtium seeds

The action on tamarind seed xyloglucan of the pure, xyloglucan-specific endo-(1-->4)-beta-D-glucanase from nasturtium (Tropaeolum majus L.) cotyledons has been compared with that of a pure endo-(1-->)-beta-D-glucanase ('cellulase') of fungal origin. The fungal enzyme hydrolysed the polysaccharide almost completely to a mixture of the four xyloglucan oligosaccharides: [formula: see text] Exhaustive digestion with the nasturtium enzyme gave the same four oligosaccharides plus large amounts of higher oligosaccharides and higher-polymeric material. Five of the product oligosaccharides (D, E, F, G, H) were purified and shown to be dimers of oligosaccharides A to C. D (glc8xyl6) had the structure A-->A, H (glc8xyl6 gal4) was C-->C, whereas E (glc8xyl6gal), F (glc8xyl6gal2) and G (glc8xyl6gal3) were mixtures of structural isomers with the appropriate composition. For example, F contained B2-->B2 (30%), A-->C (30%), C-->A (20%), B2-->B1 (15%) and others (about 5%). At moderate concentration (about 3 mM) oligosaccharides D to H were not further hydrolysed by the nasturtium enzyme, but underwent transglycosylation to give oligosaccharides from the group A, B, C, plus higher oligomeric structures. At lower substrate concentrations, hydrolysis was observed. Similarly, tamarind seed xyloglucan was hydrolysed to a greater extent at lower concentrations. It is concluded that the xyloglucan-specific nasturtium-seed endo-(1-->4)-beta-D-glucanase has a powerful xyloglucan-xyloglucan endo-transglycosylase activity in addition to its known xyloglucan-specific hydrolytic action. It would be more appropriately classified as a xyloglucan endo-transglycosylase. The action and specificity of the nasturtium enzyme are discussed in the context of xyloglucan metabolism in the cell walls of seeds and in other plant tissues.

Action of a pure xyloglucan endo‐transglycosylase (formerly called xyloglucan‐specific endo‐(1‐4)‐β‐d‐glucanase) from the cotyledons of germinated nasturtium seeds

SummaryThe action on tamarind seed xyloglucan of the pure, xyloglucan‐specific endo‐(1→4)‐β‐D‐glucanase from nasturtium (Tropaeolum majus L.) cotyledons has been compared with that of a pure endo‐(1→4)‐β‐D‐glucanase (‘cellulase’) of fungal origin. The fungal enzyme hydrolysed the polysaccharide almost completely to a mixture of the four xyloglucan oligosaccharides:image Exhaustive digestion with the nasturtium enzyme gave the same four oligosaccharides plus large amounts of higher oligosaccharides and higher‐polymeric material. Five of the product oligosaccharides (D,E,F,G,H) were purified and shown to be dimers of oligosaccharides A to C. D (glc8xyl6) had the structure A→A, H (glc8xyl6gal4) was C→C, whereas E (glc8xyl6gal), F (glc8xyl6gal2) and G (glc8xyl6gal3) were mixtures of structural isomers with the appropriate composition. For example, F contained B2→B2 (30%), A→C (30%), C→A (20%), B2B1 (15%) and others (about 5%). At moderate concentration (about 3 mM) oligosaccharides D to H were not further hydrolysed by the nasturtium enzyme, but underwent transglycosylation to give oligosaccharides from the group A, B, C, plus higher oligomeric structures. At lower substrate concentrations, hydrolysis was observed. Similarly, tamarind seed xyloglucan was hydrolysed to a greater extent at lower concentrations. It is concluded that the xyloglucan‐specific nasturtium‐seed endo‐(1→4)‐β‐D‐glucanase has a powerful xyloglucan‐xyloglucan endo‐transglycosylase activity in addition to its known xyloglucan‐specific hydrolytic action. It would be more appropriately classified as a xyloglucan endo‐transglycosylase. The action and specificity of the nasturtium enzyme are discussed in the context of xyloglucan metabolism in the cell walls of seeds and in other plant tissues.

Cleavage of xyloglucan by nasturtium seed xyloglucanase and transglycosylation to xyloglucan subunit oligosaccharides

Oligosaccharide subunits were prepared from xyloglucan (XG) by partial hydrolysis with cellulase and added back at micro- to millimolar concentrations to XG in the presence of nasturtium seed xyloglucanase (XG-ase). The oligosaccharides (0.2 m m) stimulated the capacity of this XG-ase to reduce the viscosity of XG solutions by 10- to 20-fold. Purification and fractionation of seed XG-ase activity by gel permeation fast protein liquid chromatography produced a single peak that was much more active in the presence than absence of added XG oligosaccharide. [ 14C]Fucose-labeled XG nonasaccharide was synthesized by pea fucosyltransferase and shown to be incorporated into polymeric XG in the presence of seed XG-ase without the net production of new reducing chain ends, even while the loss of XG viscosity and XG depolymerization were enhanced. It is concluded that in vitro seed XG-ase can transfer cleavage products of XG to XG oligosaccharides via endotransglycosylation reactions, thereby reducing XG M r without hydrolysis. Since this is the only XG-cleaving enzyme that develops in nasturtium seeds during germination, it may be that its transglycosylase and hydrolase capacities are both necessary to account for the rapid and complete depolymerization of XG that takes place.

Enzyme-gold cytochemistry of seed xyloglucans using two xyloglucan-specific hydrolases. Importance of prior heat-deactivation of the enzymes

Two pure, homogeneous xyloglucan-hydrolyzing enzymes from germinated nasturtium seeds have been used to localize xyloglucans specifically in seed cell walls. The enzymes, a novel endo (1----4)-beta-D-glucanase which shows absolute specificity towards xyloglucans and a beta-D-galactosidase which is capable of removing galactosyl residues from polymeric xyloglucans, were used to stabilize gold sols. The complexes were applied to ultrathin sections of nasturtium (Tropaeolum majus L) and tamarind (Tamarindus indica L) seeds. The gold complexes prepared from the active enzyme proteins retained enzyme activity, and such complexes gave extremely weak section-labelling or no labelling at all. When the enzymes were subjected to heat-deactivation before being used to stabilize the gold sols, gold complexes were obtained which lacked enzyme activity, but which gave strong, specific labelling of xyloglucans in ultrathin sections. The specificity of the labelling was checked by substrate-competition, by pretreatment of sections with the active and heat-denaturated enzymes and by comparing the labelling of xyloglucan-containing storage cells with other cell types in the same section. The labelling was maximal at the pH which was optimal for the active enzyme. We conclude that the enzyme-gold complexes which retain high activity against the substrate to be localized are likely to be unsuitable as cytochemical probes because they may cause in situ substrate modification. In the case of the enzyme complexes described here the specific localization obtained with the gold complexes prepared from heat deactivated enzymes may be attributable to the retention by the heat-treated enzymatically-inactive proteins of substrate recognition.(ABSTRACT TRUNCATED AT 250 WORDS)