Synthesis of the cyanogenic beta-glucosidase, linamarase, in white clover

The beta-glucosidase, linamarase, which specifically hydrolyzes cyanogenic substrates, linamarin and lotaustralin, in white clover, is synthesized in the early stages of leaf and seedling development in genetically competent plants. Plants, from natural populations, possessing at least one Li allele synthesize linamarase but plants with only li alleles do not, nor do they produce inactive but antigenically related linamarase. Linamarase is known to be a mannosyl glycoprotein, which in its active form is a dimer, with a subunit size of 62,000 Mr. We demonstrate that the antibiotic tunicamycin, which prevents N-acetyl-asparagine linked glycosylation, reduces in vivo synthesis of linarmarase. In vitro translation of mRNA from a Li Li plant yields a 59,000 Mr immunoprecipitated linamarase polypeptide which is modified to a 62,000 Mr product by the addition of dog pancreas microsomes. No anti-linamarase immunoprecipitable product is obtained from the in vitro translation products of mRNA from a li li plant.     

Characterization of cyanogenic beta-glucosidase (linamarase) from cassava (Manihot esculenta Crantz)

Linamarase (EC 3.2.1.21) was purified from cassava petiole, stem, and root cortex by ammonium sulfate precipitation, column chromatography on Sepharose 6B, and chromatofocusing. The last step resolved the enzyme from each source into three forms with pI values of 4.3, 3.3, and 2.9. Each form was found to be oligomeric, consisting of one kind of subunit, Mr 63,000. The major isozyme with a pI of 4.3 from petiole showed a Km for linamarin of 0.6 mM and possessed both beta-glucosidase and beta-fucosidase activities. The former was sensitive to inhibition by delta-gluconolactone, isopropyl-beta-D-thioglucoside, and HgCl2, whereas the latter was inhibited by Tris ion.     

Purification and properties of beta-D-glucosidase (linamarase) from the butter bean, Phaseolus lunatus

A beta-D-glucosidase (linamarase) was purified 11,700-fold from the butter bean, Phaseolus lunatus L., by means of successive procedures including extraction, ammonium sulfate fractionation, acetone treatment, and chromatographies on CM-Sephadex, DEAE-Sephadex, and Sephadex G-200. The final preparation gave a single protein band on both disc polyacrylamide gel electrophoresis and SDS-polyacrylamide gel electrophoresis. In spite of its electrophoretic purity, the final enzyme preparation showed four glycosidase activities; beta-D-glucosidase, beta-D-galactosidase, beta-D-fucosidase, and beta-D-xylosidase. The molecular weight of the enzyme was determined to be 124,000 +/- 9,000 by Sephadex G-200 gel filtration, and 59,000 +/- 2,400 by SDS-disc gel electrophoresis. The enzyme showed a pH optimum in the range of 5.1 to 6.0 with p-nitrophenyl beta-D-glucoside, 4-methylumbelliferyl beta-D-glucoside, and linamarin. Among natural substrates containing a beta-glucosyl terminal, linamarin, prunasin, and salicin were hydrolyzed by the enzyme from butter beans, but amygdalin, cellobiose, gentiobiose, and laminarin were hardly hydrolyzed.     

Spectrophotometer-aided evaluation of cyanogenic potential in white clover (Trifolium repens L.)

In comparison with ordinary methods of colorimetric evaluation of cyanogenic potential based on visual evaluation of the alkaline picrate reaction, a spectrophotometer-aided method could be more accurate (since it determines the exact amount of hydrogen cyanide released by the plant material), and less time-consuming as it can be performed on bulk material rather than on a number of individual plants. Ten white clover populations were evaluated by a spectrophotometer-aided method and by two visual evaluation criteria. All methods indicated the presence of large variation between populations. Visual methods gave almost identical results and allowed only for the distinction between cyanogenic and substantially acyanogenic populations. The results were only moderately consistent with those obtained by the spectrophotometer-aided method, which could detect the presence of variation also between cyanogenic populations. The effects of various incubation times (from 4 to 48 h) and of the addition of β-glucosidase on hydrogen cyanide release were also investigated. Comparable results for ranking of populations could be obtained over a range of incubation times, but at least 24 h were needed for a reliable estimation of the hydrogen cyanide produced by plants. The addition of enzyme did not increase the released cyanide. The effect of season and/or conditions of evaluation was marked on mean cyanogenic potential but limited on ranking of populations. Copyright © 2000 John Wiley & Sons, Ltd.     

A molecular and biochemical analysis of the structure of the cyanogenic beta-glucosidase (linamarase) from cassava (Manihot esculenta Cranz).

The cyanogenic beta-glucosidase (linamarase) of cassava is responsible for the first step in the sequential break-down of two related cyanoglucosides. Hydrolysis of these cyanoglucosides occurs following tissue damage and leads to the production of hydrocyanic acid. This mechanism is widely regarded as a defense mechanism against predation. A linamarase cDNA clone (pCAS5) was isolated from a cotyledon cDNA library using a white clover beta-glucosidase heterologous probe. The nucleotide and derived amino acid sequence is reported and five putative N-asparagine glycosylation sites are identified. Concanavalin A affinity chromatography and endoglycosidase H digestion demonstrate that linamarase from cassava is glycosylated, having high-mannose-type N-asparagine-linked oligosaccharides. Consistent with this structure and the extracellular location of the active enzyme is the identification of an N-terminal signal peptide on the deduced amino acid sequence of pCAS5.     

An investigation of the substrate preference of white sturgeon (Acipenser transmontanus) eleutheroembryos

Fifty white sturgeon (Acipenser transmontanus) eleutheroembryos (average size 0.17 g) were placed onto each of four quadrants (0.45 m² quadrant⁻¹; 200 fish tank⁻¹) of different sized substrates in four circular tanks (approximately 562 L). Each of three quadrants had a different size substrate and the fourth quadrant was left bare. We used one replicate of smaller size substrates (0.5–11.9 mm) and one replicate of larger size substrates (21.7–88 mm). It was found that the white sturgeon eleutheroembryos preferred substrate with an average size (longest diameter) of 12 mm (11.9) in the smaller substrate range and 22 mm (21.7) in the larger substrate range. These data improve our knowledge of white sturgeon early life history, and if confirmed in the wild can be used to protect areas that are crucial for white sturgeon recruitment and survival.     

Resistance of red and white clover cultivars to clover rot (Sclerotinla trifoliorum)

Various techniques for assessing the resistance of clover cultivars to clover rot have been investigated, including counting sclerotia around infected plants, measuring lengths of healthy and diseased rows and the use of a key to assess symptom expression on individual plants. The latter was found to be the most suitable under field conditions. Of fifty-one red clover cultivars tested, nineteen were found to possess some level of resistance. Clover rot appears to be an increasing problem with white clover crops but tests have indicated that resistant cultivars are available.     

Nodulation of white clover (Trifolium repens) in the absence ofRhizobium

Summary Spontaneous nodules developed on the roots of white clover (Trifolium repens cv. Ladino) in the absence ofRhizobium. A small subpopulation of uninoculated clover plants (0.2%) exhibited white, single-to-multilobed elongated structures on their root systems when grown without fixed nitrogen. Clonal propagation using aseptic stolons confirmed the genetic stability of the observation. Few if any viable bacteria of unknown origin were recovered from surfacesterilized structures. Nodule contents were incapable of eliciting nodulation. Histological observations showed that these structures possessed all the characteristic features of indeterminate nodules, such as active meristem, cortex, endodermal layer, vascular strands, and a central zone with parenchyma cells. Infection threads, intercellular or intracellular bacteria were absent. Instead, numerous starch grains were observed in the central zone, a feature absent in normal nitrogen-fixing nodules. Our observation broadens the concept of spontaneous nodulation, believed to be restricted to alfalfa (Medicago sativa), to other legumes, and suggests a degree of generality among indeterminately nodulated legumes displaying natural heterozygosity.     

Kinetic studies on the broad-specificity beta-D-glucosidase from pig kidney.

A broad-specificity beta-D-glucosidase from pig kidney cortex was isolated and purified to homogeneity by a rapid purification procedure. The pI (5.14 +/- 0.05), Mr (59,000 +/- 2000) and specific activities with several p-nitrophenyl glycosides (galactopyranoside, glucopyranoside, arabinopyranoside, xylopyranoside) were comparable with those published previously for cytoplasmic beta-D-glucosidase from other sources and organs. Mixed-substrate experiments and inhibition studies with glucono-(1----5)-lactone revealed that a single active centre, containing one catalytic site and one saccharide-binding site, was responsible for the splitting of all four synthetic substrates. Inhibition experiments with substrate analogues demonstrated that (i) the major binding determinant of the glycosides was the aglycone moiety, (ii) an anionic side chain of the enzyme (probably a carboxy group) interacted with the glycosidic linkages and (iii) the properties of the aglycone significantly influenced the binding of the carbohydrate moiety. The inhibition constants of the p-nitrothiophenyl derivatives were in good agreement with the Km values of the corresponding substrates. Therefore the Michaelis constants could be regarded as true equilibrium constants (Ks). The 'three-point-attachment model' of the substrate splitting, proposed by Daniels [(1983) Ph.D. Dissertation, University of Pittsburgh] for the analogous liver enzyme, was applicable for beta-D-glucosidase from pig kidney too. The possible nature of the 'attachments' is discussed.     

Kinetic characterization of the substrate specificity and mechanism of mushroom tyrosinase.

This paper reports a quantitative study of the effect of ring substituents in the 1-position of the aromatic ring on the rate of monophenol hydroxylation and o-diphenol oxidation catalyzed by tyrosinase. A possible correlation between the electron density of the carbon atom supporting the oxygen from the monophenolic hydroxyl group and the V Mmax values for each monophenol was found. In the case of o-diphenols the same effect was observed but the size of the side-chain became very important. NMR studies on the monophenols justified the sequence of the V Mmax values obtained. As regards the o-diphenols, on the other hand, only a fair correlation between NMR and V Dmax values was observed due to the effect of the molecular size of the ring substituent. From these data, it can be concluded that the redox step (k33) is not the rate-determining step of the reaction mechanism. Thus, the monophenols are converted into diphenols, but the order of specificities towards monophenols is different to that of o-diphenols. The rate-limiting step of the monophenolase activity could be the nucleophilic attack (k51) of the oxygen atom of the hydroxyl group on the copper atoms of the active site of the enzyme. This step could also be similar to or have a lower rate of attack than the electrophilic attack (k52) of the oxygen atom of the active site of oxytyrosinase on the C-3 of the monophenolic ring. However, the rate-limiting step in the diphenolase activity of tyrosinase could be related to both the nucleophilic power of the oxygen atom belonging to the hydroxyl group at the carbon atom in the 3-position (k32) and to the size of the substituent side-chain. On the basis of the results obtained, kinetic and structural models describing the monophenolase and diphenolase reaction mechanisms for tyrosinase are proposed.     

In-situ localization of cyanogenic β-glucosidase (linamarase) gene expression in leaves of cassava (Manihot esculenta Cranz) using non-isotopic riboprobes

The release of hydrogen cyanide (cyanogenesis) from damaged plant tissue depends upon the sequential action of a β-glucosidase and an α-hydroxynitrilase on cyanoglucosides. The non-isotopic digoxigenin labelling system was used to visualize the presence of cyanogenic β-glucosidase (linamarase) mRNA in cells of young leaves of Manihot esculenta Cranz (cassava). Strong hybridization to antisense riboprobes produced from the cDNA clone pCAS5, indicates localization of linamarase gene expression in laticifers (latex vessels). This is supported by the demonstration of linamarase mRNA in exuded latex. In contrast, in-situ localization of the control gene pGLF4, showed expression in all leaf mesophyll cells. High levels of linamarase activity were demonstrated in the latex of leaf petioles and this activity was shown to be dependent on the presence of attached leaflets. Assays of α-hydroxynitrilase activity in exuded latex and whole leaves shows that, unlike linamarase, this enzyme is present at very low levels in latex and must be located elsewhere in the leaf.     

Cyanoglucoside biosynthesis in white clover (Trifolium repens)

l-valine-(U-¹⁴C), isobutyraldoxime-(U-¹⁴C) and 2-hydroxyisobutyraldoxime-(1,3-¹⁴C) were fed to white clover shoots which contained or lacked cyanoglucoside. Labelling results indicated that plants which were unable to synthesize linamarin from these precursors lacked the ability to glucosylate 2-hydroxyisobutyronitrile. When l-valine-(U-¹⁴C) was fed to cyanoglucoside-producing plants in the presence of cold isobutyraldoxime, radioactivity could be trapped in this compound. No radioactivity was trapped in isobutyraldoxime fed to non-cyanoglucoside-producing shoots demonstrating that this phenotype also lacks the ability to convert valine into the aldoxime. This suggests that the non-cyanoglucoside-producing plants have at least two steps in the biosynthesis of linamarin missing. Differences between the linamarin biosynthetic enzymes of white clover and flax were demonstrated using the inhibitor O-methylthreonine and by the failure of cyanoglucoside-producing white clover shoots to synthesize linamarin from fed 2-hydroxyisobutyraldoxime.     

The inheritance of floral characters in white clover (Trifolium repens)

A diallel cross was carried out among seven varieties of white clover to investigate the inheritance of some of those characters which affect potential seed yield. Peduncle length and number of seeds/floret have low narrow-sense heritabilities, while peduncle length displays the added complications of heterozygosity and non-allelic interactions. Consequently, improvements in these characters will be difficult. Seed number m^-2 is a composite character which apparently exhibits heterozygosity. Higher seed yields will therefore be obtained by selection for its component characters, particularly floret number and floral density, both of which are apparently inherited in a comparatively straightforward manner. These results are discussed in the context of improving the potential seed yield of white clover.