Gordon Maclachlan

Endo-1,4-[beta]-Glucanase, Xyloglucanase, and Xyloglucan Endo-Transglycosylase Activities Versus Potential Substrates in Ripening Tomatoes

In ripening fruits of tomato (Lycopersicon esculentum L. var 83-G-38), the amounts of cellulose and xyloglucan (XG) remained constant during tissue softening, but the relative molecular weight (Mr) of XG decreased markedly and the Mr of cellulose declined slightly. These changes could have been due to activities of non-specific endo-1,4-[beta]-glucanases and/or buffer-soluble XG endo-transglycosylase, both of which increased when tissue firmness declined most rapidly. Tomato extracts also reduced the viscosity of XG solutions, especially in the presence of added XG oligosac-charides. This depolymerizing (XGase) capacity differed from [beta]-glucanase and XG transglycosylase activity (a) by being almost entirely buffer insoluble, and (b) by declining precipitously during fruit softening. Although it disappeared from ripe fruit, XGase may have functioned in promoting wall loosening at earlier stages of fruit development when its activity was highest. By contrast, during aging of fruit in the ripening-inhibited mutant rin there was no change in Mr of XG or cellulose, and activities of [beta]-glucanases and XG transglycosylase were lower than in wild-type tomato. Nevertheless, some softening of the fruit did take place over time and XG amounts declined, possibly because high XGase activity was maintained in the mutant, unlike in wild-type fruit.

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 mM) 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.

Stimulation of pea 1,4-β-glucanase activity by oligosaccharides derived from xyloglucan

Abstract Oligosaccharide fragments prepared by enzymic digestion of pea xyloglucan at micromolar concentrations exhibited marked stimulatory effects on pea endo-1,4-β-glucanase activity during viscometric assays with xyloglucan as the substrate. The nonasaccharide repeating subunit (Glc 4 Xyl 3 GalFuc) at 200μ m evoked a 7–10-fold stimulation of β-glucanase activity, and the heptasaccharide subunit (Glc 4 Xyl 3 ) at 200μ m evoked a 4–5-fold stimulation. The stimulatory effect was substrate- and enzyme-specific; e.g. , it could not be detected when O -(carboxymethyl)cellulose was used as the substrate or when endo-1,4-β-glucanase from Trichoderma was tested with xyloglucan as the substrate.

Sugar levels in the pea epicotyl: Regulation by invertase and sucrose synthetase

Abstract In meristematic regions of the pea epicotyl (plumule and hook), fructose was the predominant sugar (about 1 per cent fr. wt.), sucrose was also present (0·4 per cent) but glucose was barely detectable. In adjacent regions of internode where cell expansion occurred, glucose became the main sugar (1 per cent) and levels of fructose and sucrose declined. None of these sugars was absorbed or utilized for metabolism preferentially by excised sections of any one region of the epicotyl. The distribution of various glucosidase, exo-glucanase and sugar phosphatase activities bore no clear relationship to actual hexose concentrations. However, the distribution of invertase activity was parallel to that of glucose, and sucrose synthetase was most active in regions where fructose and sucrose were concentrated. It is suggested, therefore, that the principal factors controlling sugar levels in the pea epicotyl are location and relative activities of these two enzymes, both utilizing translocated sucrose as substrate.

Changes in concentration of polymeric components in excised pea-epicotyl tissue during growth

Sections were cut from apical (0–5 mm) and basal (20–25 mm) regions of the third internode of 8-day-old etiolated pea epicotyls. They were incubated for 48 h on water or 24 h on 2% [14C]sucrose. Measurements were made of: (a)_growth in length, fresh and dry weight; (b) changes in concentration of protein, nucleic acid and wall components; and (c) incorporation of radioactivity into polysaccharide. The main results were: (1) Apical but not basal sections grew in length and fresh weight. Cell-wall elasticity decreased during incubation. (2) Dry weight of apical sections fell during growth on water due to loss of protein, nucleic acid and a wall fraction containing glucose and galactose which was soluble in hot 1 N HCl. Pectic acid and pentosan levels remained constant. Lignin and cellulose increased. In basal sections, pectic acid was the only component that decreased in concentration. (3) Those wall materials which were lost from sections incubated on water showed metabolic turnover when radioactive sucrose was supplied. Ways in which growt may depend on the polymer metabolism which accompanies it are discussed. Formation of crystalline from amorphous cellulose appears to be one reaction which occurs specifically during cell elongation.

Solubilization and properties of GDP-fucose : xyloglucan 1,2-α-L-fucosyltransferase from pea epicotyl membranes

GDP-fucose:xyloglucan 1,2-alpha-L-fucosyltransferase from pea (Pisum sativum) epicotyl microsomal membranes was readily solubilized by extraction with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps). When using GDP-[14C]fucose as fucosyl donor and tamarind xyloglucan (XG) as acceptor, maximum activation was observed at 0.3% (w/v) Chaps and the highest yield of solubilized activity at 0.4%. The reaction product was hydrolyzed by Trichoderma cellulase to yield labeled oligosaccharides that peaked on gel permeation chromatography at the same elution volume as pea XG nona- and decasaccharide subunits. The apparent Km for fucosyl transfer to tamarind XG by the membrane-bound or solubilized enzyme was about 80 microM GDP-fucose. This was 10 times the apparent Km for fucosyl transfer to endogenous pea nascent XG. Optimum activity was between pH 6 and 7, and the isoelectric point was close to pH 4.8. The solubilized enzyme showed no requirement for, or stimulation by, added cations or phospholipids, and was stable for several months at -70 degrees C. Solubilization and gel permeation chromatography on columns of Sepharose CL-6B enriched the specific activity of the enzyme by about 20-fold relative to microsomes. Activity fractionated on columns of CL-6B with an apparent molecular weight of 150 kDa. The solubilized fucosyltransferase was electrophoresed on nondenaturing polyacrylamide slab gels containing 0.02% (w/v) tamarind XG, and its activity located by incubation in GDP-[14C]fucose, washing, and autoradiographing the gel. A single band of labeled reaction product appeared with an apparent molecular weight of 150 kDa.

Xyloglucan Galactosyl- and Fucosyltransferase Activities from Pea Epicotyl Microsomes

Microsomal membranes from growing tissue of pea (Pisum sativum L.) epicotyls were incubated with the substrate UDP-[14C]galactose (Gal) with or without tamarind seed xyloglucan (XG) as a potential galactosyl acceptor. Added tamarind seed XG enhanced incorporation of [14C]Gal into high-molecular-weight products (eluted from columns of Sepharose CL-6B in the void volume) that were trichloroacetic acid-soluble but insoluble in 67% ethanol. These products were hydrolyzed by cellulase to fragments comparable in size to XG subunit oligosaccharides. XG-dependent galactosyltransferase activity could be solubilized, along with XG fucosyltransferase, by the detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate. When this enzyme was incubated with tamarind (Tamarindus indica L.) seed XG or nasturtium (Tropaeolum majus L.) seed XG that had been partially degalactosylated with an XG-specific [beta]-galactosidase, the rates of Gal transfer increased and fucose transfer decreased compared with controls with native XG. The reaction products were hydrolyzed by cellulase to 14C fragments that were analyzed by gel-filtration and high-performance liquid chromatography fractionation with pulsed amperometric detection. The major components were XG subunits, namely one of the two possible monogalactosyl octasaccharides (-XXLG-) and digalactosyl nonasaccharide (-XLLG-), whether the predominant octasaccharide in the acceptor was XXLG (as in tamarind seed XG) or XLXG (as in nasturtium seed XG). It is concluded that the first xylosylglucose from the reducing end of the subunits was the Gal acceptor locus preferred by the solubilized pea transferase. These observations are incorporated into a model for the biosynthesis of cell wall XGs.

Xyloglucan oligosaccharide α-l-fucosidase activity from growing pea stems and germinating nasturtium seeds

[14C]Fucose-labelled xyloglucan (XG) was synthesized from tamarind seed XG by incubating it with GDP-[14C]fucose plus solubilized pea fucosyltransferase, and [14C]fucose-labelled XG nonasaccharide was prepared from the parent hemicellulose by partial hydrolysis with fungal cellulase. alpha-L-Fucosidase activity was readily detected in crude enzyme extracts of growing regions of etiolated pea stems (Pisum sativum) and in cotyledons of germinating nasturtium seedlings (Tropaeolum majus) using the fucosylated XG-nonasaccharide as substrate. Both enzymes showed little activity against intact fucosylated XG and they were totally inactive against p-nitrophenyl-alpha-L-fucoside. Auxin treatment of pea stems, which greatly increased the activity of endo-1,4-beta-glucanases that hydrolyse XG in apical growing regions, failed to result in a similar increase in XG-nonasaccharide alpha-fucosidase activity. However, germination of nasturtium seed, which resulted in a large increase in endo-1,4-beta-glucanase (XG-ase) activity in the cotyledons, was accompanied by comparable increases in XG-alpha-fucosidase activity.

Synthesis of wall glucan from sucrose by enzyme preparations from Pisum sativum

Abstract Radioactive sucrose, supplied through the cut base to Pisum sativum epicotyls, was transported to the growing apex (plumule and hook) and used there for the synthesis mainly of uridine diphosphoglucose (UDP- glucose), fructose and cell wall glucan. Enzyme extracts of the apical tissue contained sucrose synthetase activity which was freely reversible, i.e. formed UDP-glucose and fructose from sucrose (pH optimum = 6·6 for the cleavage reaction, K m for sucrose = 63 mM). Particulate fractions of the same tissue contained a β-glucan synthetase which utilized UDP-glucose for formation of alkali-soluble and -insoluble products (pH optimum = 8·4, K m for UDP-glucose = 1·9 mM). Values for V max and yields of these two synthetase activities were sufficient to account for observed rates of cellulose deposition during epicotyl growth (15–25 μg/hr/epicotyl). When soluble pea enzyme was supplied with sucrose and UDP at pH 6·6 and then the preparation was supplemented with particles bearing β-glucan synthetase at pH 8·4, the glucose moiety of sucrose was converted to glucan in vitro . The results indicate that it is feasible for these synthetases to co-operate in vivo to generate β-glucan for expanding cell walls.

Effects of indoleacetic acid on intracellular distribution of β-glucanase activities in the pea epicotyl

Abstract β-1,4-Glucanase (cellulase) and β-1,3-glucanase activities were estimated in various subfractions of the growing region of decapitated Pisum sativum epicotyls. Treatment of the epicotyl apex with the hormone indoleacetic acid (IAA) caused total and specific cellulase activity to increase, first and most markedly, in a microsomal fraction containing membrane-bound ribosomes and eventually in fractions containing soluble enzyme and wall material. The responses did not occur in the presence of actinomycin D or during growth after treatment with other hormones. In the absence of IAA, cellulase activity disappeared within a few days from microsomal and wall fractions. It is concluded that cellulase is synthesized via an IAA-dependent mechanism and is inactivated extracellularly. In the same system, β-1,3-glucanase activity increased greatly with time but microsomes failed to show preferential enrichment at any stage of development and IAA treatment failed to affect the increase unilaterally.