Glucomannan synthesis in pea epicotyls: the mannose and glucose transferases.

Membrane fractions and digitonin-solubilized enzymes prepared from stem segments isolated from the third internode of etiolated pea seedlings (Pisum sativum L. cv. Alaska) catalyzed the synthesis of a beta-1,4-[14C]mannan from GDP-D-[U-14C]-mannose, a mixed beta-1,3- and beta-1,4-[14C]glucan from GDP-D-[U-14C]-glucose and a beta-1,4-[14C]-glucomannan from both GDP-D-[U-14C]mannose and GDP-D-[U-14C]glucose. The kinetics of the membrane-bound and soluble mannan and glucan synthases were determined. The effects of ions, chelators, inhibitors of lipid-linked saccharides, polyamines, polyols, nucleotides, nucleoside-diphosphate sugars, acetyl-CoA, group-specific chemical probes, phospholipases and detergents on the membrane-bound mannan and glucan synthases were investigated. The beta-glucan synthase had different properties from other preparations which bring about the synthesis of beta-1,3-glucans (callose) and mixed beta-1,3- and beta-1,4- glucans and which use UDP-D-glucose as substrate. It also differed from xyloglucan synthase because in the presence of several concentrations of UDP-D-xylose in addition to GDP-D-glucose no xyloglucan was formed. Using either the membrane-bound or the soluble mannan synthase, GDP-D-glucose acted competitively in the presence of GDP-D-mannose to inhibit the incorporation of mannose into the polymer. This was not due to an inhibition of the transferase activity but was a result of the incorporation of glucose residues from GDP-D-glucose into a glucomannan. The kinetics and the composition of the synthesized glucomannan depended on the ratio of the concentrations of GDP-D-glucose and GDP-D-mannose that were available. Our data indicated that a single enzyme has an active centre that can use both GDP-D-mannose and GDP-D-glucose to bring about the synthesis of the heteropolysaccharide.

Spatial and temporal patterns of transcription of a wound-induced gene in potato.

Transcriptional fusions between the gene encoding win2 from potato and the reporter gene encoding beta-glucuronidase (GUS) have been used to study the spatial and temporal patterns of wound induced gene activity in transgenic potato and tobacco plants. Gene fusions containing a full length win2 promoter were found to be correctly regulated in response to mechanical wounding in transgenic potato, but not in the heterologous host, tobacco. Sequences greater than 560 bp upstream of the transcription start site of win2 were shown to be important for wound inducibility. The dramatic induction of GUS activity detected using fluorometric assays of extracts of wounded and aged leaves of several independent win2--GUS transformants was consistent with the kinetics of win2 mRNA accumulation. Histochemical analysis of wounded leaves showed that transcription first occurred in cells immediately adjacent to the wound, and was then progressively induced in cells associated with the vascular system at a distance from the wound site. In tubers, a localized response to wounding was observed, and this only spread to other parts of the tuber if it had started to sprout. It was concluded that active vascular transport was necessary for the spread of wound response. Win2--GUS fusions were also expressed as part of normal plant development, as GUS activity was detected in the developing buds and in a layer of cells associated with the lenticels of unwounded tubers.

Expression of phenylalanine ammonia-lyase gene during tracheary-element differentiation from cultured mesophyll cells ofZinnia elegans L.

Expression of the phenylalanine ammonialyase (PAL) gene during tracheary-element differentiation was studied in mesophyll cell-suspension cultures ofZinnia elegans. Dose-response curves of benzyladenine (BA) and α-naphthaleneacetic acid (NAA) were obtained for the cultures in order to achieve the highest percentage of differentiation. The optimal concentrations of BA and NAA were 0.1 mg·l(-1) and 0.06 mg·l(-1), respectively, which normally stimulated about 40% differentiation by 96 h of culture. The effects of the same ratio but different amounts of BA and NAA on tracheary-element formation have been tested and the results indicate that the absolute amounts of BA and NAA rather than the ratio of them were important for tracheary-element formation in theZinnia cultures. The cells when cultured in the presence of 0.001 mg·l(-1) of BA and 0.06 mg·l(-1) of NAA expanded and divided but did not differentiate. The level of PAL activity, synthesis of PAL protein and the level of PAL mRNA peaked during 72 to 96 h when lignin was actively deposited. This indicated that the PAL gene was temporally and preferentially expressed in association with the lignification during tracheary-element differentiation and thus it can be regarded as a molecular marker for the process.

A 1,4-ß-D-glucan-synthase system from Dictyostelium discoideum.

Particulate membrane preparations have been isolated from culminating Dictyostelium discoideum cells. The preparations incorporated glucose from uridine 5'-diphosphate-glucose into a glucose polymer or polymers. These have been shown to be homopolymers of ß-linked glucose. A high percentage (78% by methylation analysis) of the linkages formed are 1,4-linkages and a lower percentage (12%) are 1,3-linkages. The glucan-synthase complex present in the particulate membrane preparation has an apparent Km of 0.28 mM and a Vmax of 1.59 nmol·min(-1)·(mg protein)(-1). The enzyme system is dependent upon Mg(2+) and cellobiose for maximal activity, but is inhibited by millimolar levels of Ca(2+). Particulate membrane preparations were made from cells at various times during a synchronous developmental time course and demonstrated that the glucan-synthase activity appeared at the tight-aggregate stage of development.

The mucilage secreted by roots and its possible role in cell-cell recognition for the adhesion of fungal pathogens to root surfaces of Zea mays L.

The outer cells of the roots of plants secrete a mucilage which lubricates the root and keeps it moist. The mucilage is secreted from the Golgi apparatus in vesicles which fuse at the plasma membrane. In maize roots a complex of at least three polysaccharides and glycoproteins are formed, some of which have a large proportion of fucose in their composition. The synthesis of these compounds can be readily monitored because fucose can be easily identified, and especially because exogenous fucose is not catabolized but is incorporated intact into the polymers. The synthesis of the polymers seems to be initiated in the endoplasmic reticulum in conjunction with polyprenoid oligosaccharides that contain fucose. Lipid-oligosaccharides of nine sugar residues can be obtained from the membrane preparations of the root cells. These compounds are polyprenyl diphosphate derivatives. A GDP-fucose:polyprenyl phosphate transfucosylase occurs in the endoplasmic reticulum, whereas fucosyl transferase that transfers fucose to a polymer occurs mainly in the Golgi apparatus. The indirect evidence suggests that oligosaccharides of polyprenyl diphosphate compounds are transferred to proteins, elaborated in the Golgi apparatus, and large molecular weight polysaccharides are finally exported as the mucus. Part of the mucus is acidic and in some respects resembles pectin. The presence of fucose in such large quantities in maize root mucilage suggested that this might have some significance for the recognition of these plants by parasitic root fungi. The adsorption of mucilage by pathogenic fungi was investigated with two types of fungi, a highly specialized ectotrophic root-infecting fungus, e.g. Phialophora radicicola and a vascular wilt fungus capable of attacking a great variety of tissues, e.g. Fusarium moniliforme. The adsorption of radioactively labelled and fluorescently labelled polymers by the pathogenic fungi was investigated. The character and proportion of fungal surfaces present in vitro were standardised by the production and semi-synchronous germination of populations of conidia. Changes in appearance of fungal walls, present before and after germination, were examined ultrastructurally. There was polyanionic material on hyphal but less on conidial surfaces of the ectotrophic root-infecting fungi. In contrast this material was present to similar extents on both hyphal and conidial surfaces of F. moniliforme.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification and purification of a nuclease from Zinnia elegans L.: a potential molecular marker for xylogenesis.

A single-strand specific nuclease was identified during a particular stage of a defined cellular differentiation pathway characteristic of xylem development. Using a hormone-inducible system in which cultured mesophyll cells of Zinnia elegans differentiated to xylem cells in synchrony, the enzymatic activity on single-stranded (ss) DNA was highest during the maturation phase of differentiation. Nondifferentiating cells contained little of this activity throughout a similar course of culture. After electrophoresis of extracts from differentiating cells, a 43-kilodalton (kDa) polypeptide was detected by its activity in the gels containing either ssDNA or RNA. Lectins specific for mannose residues on glycoproteins bound to the 43-kDa nuclease and were used to distinguish it from several ribonucleases. The nuclease was purified by a two-step chromatographic procedure: a lectin-affinity column followed by a phosphocellulose column. The purified protein was determined to be a single polypeptide with a relative molecular mass of 43000 by the analysis of its mobility during sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel filtration of the native enzyme. A sensitive detection system using biotinylated-concanavalin A and avidin was demonstrated to be specific as a probe for the nuclease protein. An N-terminal amino-acid sequence was derived from 5 pmol of the enzyme. The nuclease was most active on ssDNA at pH 5.5 in the presence of Zn(2+) and dithiothreitol. The purified preparation hydrolyzed RNA and to a lesser extent, native DNA. It digested closed circular duplex DNA by introducing a single endonucleolytic cleavage followed by random hydrolysis. During the induced pathway of synchronous differentiation in Zinnia the 43-kDa nuclease rapidly increased just prior to the onset of visibly differentiated features, and developed to a maximum level during xylem cell maturation. At this time a similar but slightly smaller nuclease appeared and became dominant as differentiation continued, and subsequently both enzymes decayed. After autolysis, a nuclease of about 37 kDa was found together with the 43-kDa enzyme in the culture medium. Complementing these analyses was the examination of the tissue distribution of the 43-kDa enzyme in Zinnia and other dicotyledonous plants, which also indicated an invivo role of the nuclease in autolysis, the terminal stage of vascular differentiation in plants. The Zinnia nuclease is therefore a potential marker for xylogenesis.

Use of antisera to localize callose, xylan and arabinogalactan in the cell-plate, primary and secondary walls of plant cells.

Antibodies to cellobiose, L-arabinopyranose, L-arabinofuranose, D-galactose, oligosaccharides containing ß1→4 xylose, oligosaccharides containing ß1→4 glucose, and oligosaccharides containing ß1→3 glucose have been raised in rabbits. The antisera have been characterized to show the specificity of binding to particular polysaccharides. They have been used for immunocytology using the electron microscope to locate the polymers in dividing and differentiating cells of bean (Phaseolus vulgaris L.) root, bean callus tissue and cells of Zinnia elegans L. in vitro. Arabinogalactans have been shown to be present in the cell-plate and primary walls but not in secondary thickening. Xylan as distinct from xyloglucan was found in the primary walls but not in the cell-plate. It was present in large amounts in the secondary thickening. Callose was found in the cell plate and also in the young growing wall. In the wall it was specifically located at the plasmodesmata. The use of the antibody against L-arabinofuranose enabled a specific organelle to be detected which was membranous and which occurred within the cytoplasm and also within the vacuole of the cells. Membranes carrying polymers containing L-arabinofuranose were also found in layers just under the plasmamembrane.

A membrane-bound enzyme complex synthesising glucan and glucomannan in pine tissues.

Particulate membrane preparations isolated from cambial cells and differentiating and differentiated xylem cells of pine (Pinus sylvestris L.) trees synthesised [(14)C]glucans using either guanosine 5'-diphosphate (GDP)-D-[U-(14)C]glucose or uridine 5'-diphosphate (UDP)-D-[U-(14)C]glucose as glycosyl donors. Although these glucans had ß-(1→3) and ß-(1→4) linkages in an approximate ratio 1:1, the distribution of the linkages in the glucan synthesised from GDP-D-glucose was different from that synthesised from UDP-D-glucose. The synthesis of the mixed ß-(1→3) and ß-(1→4) glucan from GDP-D-[U-(14)C]glucose was changed to that of ß-(1→4) glucomannan in the presence of increasing concentrations of GDP-D-mannose. The glucan formed from UDP-D-[U-(14)C]glucose was not affected by any concentration of GDP-D-mannose. The membrane preparations epimerized GDP-D-glucose to GDP-D-mannose; however, the low amount of GDP-D-mannose formed was not incorporated into the polymer becaus the affinity of the synthase for GDP-D-glucose was much greater than that for GDP-D-mannose. The glucan formed from GDP-D-glucose and the glucomannan formed from GDP-D-glucose together with GDP-D-mannose were characterized. The apparent K m and V max of the glucan synthase for GDP-D-glucose were 6.38 µM and 5.08 µM·min(-1), respectively. No lipid intermediates were detected during the synthesis of either glucan or glucomannan. The results indicated that an enzyme complex for the formation of the glucomannan was bound to the membrane.

Transcription and translation of phloem protein (PP2) during phloem differentiation in Cucurbita maxima.

The synthesis of a major phloem protein, PP2, was investigated by measurement of the mRNA at various stages of phloem development in Cucurbita. Quantitative assays with immuno-electrophoresis showed that the amounts of PP2 in hypocotyls of Cucurbita seedlings increased with the age of seedlings. An increase in mRNA for PP2 during the early stages of seedling growth was also observed by immunoprecipitation of the invitro translation products of hypocotyl polyadenylated RNA. There was close timing in the variations of PP2 synthesised in vivo and in the changes in amounts of translatable PP2-mRNA during the course of seedling growth. A complementary-DNA (cDNA) library to polyadenylated RNA from hypocotyls of 3-d-old Cucurbita seedlings has been constructed. Two cDNA clones, A and B, have been identified by hybrid-release translation to be complementary to the mRNA coding for PP2. The levels of total mRNA for PP2 measured with clone A were found to increase in the first 4 d of seedling growth but decreased to lower levels in older seedlings. Regulatory controls on both transcription and modification of transcripts appeared to occur during the synthesis of PP2.

Effect of abscisic and gibberellic acids on malate synthase transcripts in germinating castor bean seeds.

Several clones complementary to malate synthase mRNA have been identified in a complementary-DNA library to mRNA from castor bean endosperm. One of these clones has been used as a probe to measure levels of transcripts during seed germination and the effects of gibberellic acid and abscisic acid on these levels have been examined.Malate synthase transcripts increased during germination and GA3 advanced their appearance in the endosperm. Exogenously applied ABA inhibited the accumulation of transcripts over a time course of germination but the addition of GA3 counteracted its inhibitory effects. The data confirmed previous reports which indicated that the action of both growth regulators was on transcript accumulation and that there is a coordinated induction of the enzymes involved in the lipid metabolism in oil seeds.

Nucleic acid (cDNA) and amino acid sequences of isocitrate lyase from castor bean.

A cDNA library was constructed to mRNA enriched for isocitrate-lyase mRNA from castor-bean (Ricinus communis var. zanzibarensis) endosperms. Nine clones for isocitrate lyase (EC 4.1.3.1) were identified. The insert of 2.2 kb from clone ICL4 was sequenced and proved to contain the entire coding region, 1731 bp, for isocitrate lyase. The amino acid sequence of isocitrate lyase was deduced from the nucleic acid sequence. By analogy with muscle aldolase a lysine residue that possibly takes part in the binding of the substrate was identified. The 3' untranslated region contained three putative polyadenylation addition signals and two direct repeats.

Cell-cell recognition of host surfaces by pathogens. The adsorption of maize (Zea mays) root mucilage by surfaces of pathogenic fungi.

The adsorption of radioactive mucilage by pathogenic fungi was shown to be dependent upon time, the composition of mucilage, the type of fungal surface (conidia, hyphae, hyphal apices), fungal species, pH and bivalent cations. All fungal adhesins were inactivated by either proteinase or polysaccharase treatments. Adsorption was not inhibited by the numberous mono-, di- and oligo-saccharides that were tested individually, but it was inhibited absolutely by several polysaccharides. This suggested that adsorption of mucilage by pathogens involved conformational and ionic interactions between plant and fungal polymers but not fungal lectins bound to sugar residues of mucilage. Several fractionation schemes showed that pathogens bound only the most acidic of the variety of polymers that comprise mucilage. There was not any absolute distinction between ability to bind radioactive mucilage and type of pathogen or non-pathogen. However, there were notable differences in characteristics of adsorption between two types of pathogen. Differences were revealed by comparison of the adsorption capacities of conidia and germinant conidia and chromatography of radioactive mucilage on germinant conidia. An ectotrophic root-infecting fungus (a highly specialized pathogen) bound a greater proportion of mucilage than did a vascular-wilt fungus (of catholic host and tissue range) with more than one class of site for adsorption. In contrast with the vascular-wilt fungus, sites for adsorption on the specialized pathogen were present solely on surfaces formed by germination.

Glucomannan-synthase activity in differentiating cells of Pinus sylvestris L.

Particulate membrane preparations have been isolated from cambial cells, and from differentiating and differentiated xylem cells of the main stem of pine trees. These preparations synthesise a ß1→4 glucomannan from guanosine 5'-diphosphate-mannose. The polysaccharide and the synthase have been characterized and the Km and Vmax for the synthase determined as 85 µM and 52.9 µM·min(-1), respectively. The enzymic activity was inhibited by the addition of guanosine 5'-diphosphate-D-glucose so that the presence of an epimerase on the particulate fraction in conjunction with the synthase probably allowed the heteropolymer to be formed with the optimal ratio of the concentrations of the nucleoside-diphosphate sugar donors. No evidence for a polyprenyl-phosphate derivative as an intermediate during the polymer synthesis was obtained. Part of the control mechanism for the deposition of the large amounts of the glucomannan during the secondary thickening of the tracheids of the vascular system is by an increase in the amount of synthase activity at the endomembrane system of the cells. This probably occurs by an increase in the amount of enzyme which is modulated by gene regulation during differentiation.

In vitro glucan synthesis by membranes of celery petioles: the role of the membrane in determining the type of linkage formed.

Glucan synthesis was achieved with an in vitro membrane fraction from the petioles of celery (Apium graveolens). The optimum conditions for maximum synthesis were established. The Km and Vmax for the enzymic system were 1.0 mM and 0.19 microM min-1 mg protein-1, respectively. Mechanical damage to the membrane fraction altered the proportion of beta-(1----3) to beta-(1----4) glucosyl linkages that were synthesized. We suggest that cellulose synthesis (beta-(1----4)-linked glucan chains) is controlled by the availability of UDP-glucose at the plasma membrane surface in conjunction with an organized relationship between the synthase system and a specifically oriented glucosyl radical acting as an acceptor held on the membrane surface. An intact membrane is therefore necessary to direct synthesis for the beta-(1----4) bond by an enzyme that is capable of transglucosylation to the secondary alcoholic groups on C-2, C-3 or C-4 of the acceptor radical. The specificity of the system is controlled by the whole enzyme complex held on the membrane.

The action of exogenous abscisic and gibberellic acids on gene expression in germinating castor beans.

Exogenously applied abscisic acid inhibits isocitrate-lyase activity of the endosperm during germination of castor-bean seeds. Amounts of isocitrate-lyase mRNA have been estimated by immunoprecipitation of in-vitro-translated polypeptide products. Exogenous abscisic acid leads to an inhibition of isocitrate lyase-mRNA accumulation. A large proportion of this effect of the growth factor may be accounted for by its action in inhibiting the overall accumulation of ribosomal RNA and total mRNA. However, the effect of abscisic acid on protein synthesis is not general, as the production of some mRNAs was stimulated. The major mRNA stored in the dry seed, coding for a 25600-Mr polypeptide that normally disappears within the first 12 h of germination, exhibited high levels in abscisic-acid-treated endosperms throughout the germination period. Three complementary DNA clones, of which two clones are complementary to isocitrate lyase, have been used to measure levels of transcripts during seed germination. The accumulation of both transcripts was inhibited by exogenous abscisic acid. The data strongly indicate that the action of abscisic acid on isocitrate lyase synthesis is either to inhibit the transcription, or to increase the transcript turnover. Exogenous gibberellic acid is able to counteract the inhibitory effects of abscisic acid.

The action of exogenous abscisic acid on malate-synthase synthesis in germinating castor-bean seeds.

The presence of 30 µM abscisic acid inhibited development of malate-synthase activity in the endosperm of germinating castor-bean seeds. Malate synthase was purified from castor-bean endosperms and an antibody to it was prepared from rabbit serum. This antibody was used to measure the amounts of malate-synthase mRNA using an in-vitro translation system. The effect of abscisic acid appeared to be greater on malate-synthase mRNA than on the bulk of mRNA, indicating some specificity of abscisic-acid action. The extent of the inhibition of malate-synthase activity and of malate-synthase mRNA accumulation were similar. This indicates that abscisic acid inhibits malate-synthase activity by lowering levels of translatable malate-synthase mRNA rather than by affecting the translation rate of this mRNA.

Stability of the complex formed between French bean (Phaseolus vulgaris) phenylalanine ammonia-lyase and its transition-state analog.

Phenylalanine ammonia-lyase forms trans-cinnamate from L-phenylalanine, and thus stands at a gateway to secondary metabolism in higher plants. L-alpha-Amino-oxy-beta-phenylpropanoic acid (L-AOPP), a very effective competitive inhibitor of this enzyme, is most probably a transition-state analog for the elimination reaction. A preparation of phenylalanine ammonia-lyase (PAL), obtained from diluted suspension cultures of French bean cells, was used to investigate the binding of this compound in vitro. After extensive dialysis, the inhibitor remained tightly bound to the enzyme unless both an increased temperature and L-phenylalanine were provided, when the spectrophotometer trace of enzyme activity gradually approached linearity. Under such optimal catalytic conditions (37 degrees C; 25 mM L-phenylalanine; pH 8.8), dissociation of the enzyme-ligand complex took place with a half-time of approx 10 min. (This is much longer than reported for the enzyme from maize.) The consequences of these findings are discussed for investigations where L-AOPP is applied in vivo. These experiments have shown that the irreversible binding of the transition-state analog under appropriate conditions (0-4 degrees C, no L-phenylalanine) gave continued protection against attack on the enzyme by an excess of borohydride. By titrating the enzyme with increasing concentrations of analog and measuring the degree of protection afforded, the active-site concentration has been estimated. The turnover number (kcat = 0.8 s-1) given by this novel approach is of the same order of magnitude as previously reported from extensive purification of enzyme from other species.