Determination of the pore size of cell walls of living plant cells.

The limiting diameter of pores in the walls of living plant cells through which molecules can freely pass has been determined by a solute exclusion technique to be 35 to 38 angstroms for hair cells of Raphanus sativus roots and fibers of Gossypium hirsutum, 38 to 40 angstroms for cultured cells of Acer pseudoplatanus, and 45 to 52 angstroms for isolated palisade parenchyma cells of the leaves of Xanthium strumarium and Commelina communis. These results indicate that molecules with diameters larger than these pores would be restricted in their ability to penetrate such a cell wall, and that such a wall may represent a more significant barrier to cellular communication than has been previously assumed.

Synthesis of Fibrils in Vitro by a Solubilized Cellulose Synthase from Acetobacter xylinum.

A digitonin-solubilized cellulose synthase was prepared from Acetobacter xylinum. When this enzyme was incubated under conditions known to lead to active synthesis of 1,4-beta-D-glucan polymer (cellulose), electron microscopy revealed that clusters of fibrils were assembled within minutes. Individual fibrils are 17 +/- 2 angstroms in diameter. Evidence that the fibrils were freshly synthesized and cellulosic in nature was their incorporation of the tritium from UDP-[(3)H]glucose (UDP, uridine 5'-diphosphate), their binding of gold-labeled cellobiohydrolase, and an electron diffraction pattern with 004, 200, and 012 reflections (characteristic of cellulose synthesized in vivo) but missing 110 and 110 reflections. The small size of the fibrils is atypical of native A. xylinum cellulose microfibrils. The fibrils synthesized in vitro resemble, in morphology and size, the fibrillar cellulose produced when A. xylinum is cultured in the presence of agents that interfere with the normal process of crystallization of the microfibrils. The solubilized enzyme unit may therefore be producing a basic fibrillar structure that, in vivo, interacts laterally with other fibrils to produce native cellulose microfibrils.

Stimulation of Callose Synthesis in Vivo Correlates with Changes in Intracellular Distribution of the Callose Synthase Activator [beta]-Furfuryl-[beta]-Glucoside.

[beta]-Furfuryl-[beta]-glucoside (FG) has been shown to be a specific endogenous activator of higher plant callose synthase (P. Ohana, D.P. Delmer, G. Volman, J.C. Steffens, D.E. Matthews, M. Benziman [1992] Plant Physiol 98: 708-715). Because glycosides such as FG are usually sequestered in vacuoles, we have proposed that activation of callose synthesis in vivo may involve a change in the compartmentation of FG and Ca2+, resulting in a synergistic activation of callose synthase. The use of suspension-cultured barley (Hordeum bulbosum L.) cells provides evidence that FG is largely sequestered in the vacuole. Furthermore, conditions that lead to induction of callose synthesis in vivo correspondingly lead to elevation of the cytoplasmic concentration of FG. These conditions include the lowering of cytoplasmic pH or elevation of cytoplasmic Ca2+. Oligogalacturonide elicitors have also been reported to cause similar changes in cytoplasmic pH and Ca2+ concentration (Y. Mathieu, A. Kurkdjian, H. Xia, J. Guern, A. Koller, M.D. Spiro, M. O'Neill, P. Albersheim, A. Darvill [1991] The Plant Journal 1: 333-343), and such an elicitor also causes an elevation in cytoplasmic FG coupled with stimulation of callose synthesis. These results support the concept that a relative redistribution of FG between cytoplasm and vacuole may be one of the components of the signal transduction pathway for elicitation of callose synthase in vivo.

In Vitro Prenylation of the Small GTPase Rac13 of Cotton.

Previous work (D.P. Delmer, J. Pear, A. Andrawis, D. Stalker [1995] Mol Gen Genet 248: 43-51) has identified a gene in cotton (Gossypium hirsutum), Rac13, that encodes a small, signal-transducing GTPase and shows high expression in the fiber at the time of transition from primary to secondary wall synthesis. Since Rac13 may be important in signal transduction pathway(s), regulating the onset of fiber secondary wall synthesis, we continue to characterize Rac13 by determining its ability to undergo posttranslational modification. In animals Rac proteins contain the C-terminal consensus sequence CaaL (where "a" can be any aliphatic residue), which is a site for geranylgeranylation (B.T. Kinsella, R.A. Erdman, W.A. Maltese [1994] J Biol Chem 266: 9786-9794). We have identified activities in developing cotton fibers that resemble in specificity the geranylgeranyl- and farnesyltransferases of animals and yeast. In addition, using prenyltransferases from rabbit reticulocytes, we show that Rac13, having a C-terminal sequence of CAFL, can serve as an in vitro substrate for geranylgeranylation but not farnesylation. However, the presence of the uncommon penultimate F residue appears to slow the rate of prenylation considerably compared with other acceptors.

The Differential Expression of Sucrose Synthase in Relation to Diverse Patterns of Carbon Partitioning in Developing Cotton Seed.

Developing cotton (Gossypium hirsutum L.) seed exhibits complex patterns of carbon allocation in which incoming sucrose (Suc) is partitioned to three major sinks: the fibers, seed coat, and cotyledons, which synthesize cellulose, starch, and storage proteins or oils, respectively. In this study we investigated the role of Suc synthase (SuSy) in the mobilization of Suc into such sinks. Assessments of SuSy gene expression at various levels led to the surprising conclusion that, in contrast to that found for other plants, SuSy does not appear to play a role in starch synthesis in the cotton seed. However, our demonstration of functional symplastic connections between the phloem-unloading area and the fiber cells, as well as the SuSy expression pattern in fibers, indicates a major role of SuSy in partitioning carbon to fiber cellulose synthesis. SuSy expression is also high in transfer cells of the seed coat facing the cotyledons. Such high levels of SuSy could contribute to the synthesis of the thickened cell walls and to the energy generation for Suc efflux to the seed apoplast. The expression of SuSy in cotyledons also suggests a role in protein and lipid synthesis. In summary, the developing cotton seed provides an excellent example of the diverse roles played by SuSy in carbon metabolism.

Sucrose synthase localizes to cellulose synthesis sites in tracheary elements.

The synthesis of crystalline cellulose microfibrils in plants is a highly coordinated process that occurs at the interface of the cortex, plasma membrane, and cell wall. There is evidence that cellulose biogenesis is facilitated by the interaction of several proteins, but the details are just beginning to be understood. In particular, sucrose synthase, microtubules, and actin have been proposed to possibly associate with cellulose synthases (microfibril terminal complexes) in the plasma membrane. Differentiating tracheary elements of Zinnia elegans L. were used as a model system to determine the localization of sucrose synthase and actin in relation to the plasma membrane and its underlying microtubules during the deposition of patterned, cellulose-rich secondary walls. Cortical actin occurs with similar density both between and under secondary wall thickenings. In contrast, sucrose synthase is highly enriched near the plasma membrane and the microtubules under the secondary wall thickenings. Both actin and sucrose synthase lie closer to the plasma membrane than the microtubules. These results show that the preferential localization of sucrose synthase at sites of high-rate cellulose synthesis can be generalized beyond cotton fibers, and they establish a spatial context for further work on a multi-protein complex that may facilitate secondary wall cellulose synthesis.

Dimethylsulfoxide as a potential tool for analysis of compartmentation in living plant cells.

Data are presented which indicate that dimethylsulfoxide (DMSO) acts selectively on the plasma membrane of cultured tobacco cells, rendering it more permeable to small molecules, while having a far smaller effect on the permeability of the vacuolar membrane. The results which support this conclusion are: (a) DMSO (5 to 10%, by volume) causes complete release of [(14)C]tryptophan newly synthesized from [(14)C]indole while causing efflux of only about 20% of the total intracellular tryptophan pool; (b) similar concentrations of DMSO do not cause substantial release from these cells of phenolic compounds or preloaded neutral red, nor of beta-cyanin from fresh beet discs; (c) kinetic studies of release of tryptophan and neutral sugars and of efflux of (86)Rb(+) show that DMSO selectively promotes rapid release of a portion of the total pool, followed by a substantially slower release of the remaining pool; (d) when tobacco cell protoplasts are incubated in the presence of 7.5% (by volume) DMSO, rapid lysis is observed concomitant with the release of intact vacuoles. These data indicate that a procedure involving a brief treatment of intact plant cells or tissues with DMSO may be used to assess the distribution of metabolites between cytoplasmic and vacuolar compartments.

The Regulatory Properties of Purified Phaseolus aureus Sucrose Synthetase.

Phaseolus aureus sucrose synthetase, purified to homogeneity, was assayed in the presence of a variety of biological compounds to test for possible regulatory effectors. The oxidized form of nicotinamide adenine dinucleotide phosphate, as well as indoleacetic acid, gibberellic acid, and pyrophosphate were found to activate the forward reaction (sucrose degradation) and inhibit the reverse reaction (sucrose synthesis). The reduced form of nicotinamide adenine dinucleotide phosphate antagonizes the effect of the oxidized form. Fructose 1-phosphate and divalent cations inhibit the forward and activate the reverse reaction. Pyrophosphate and fructose 1-phosphate are effective only in the presence of magnesium chloride. Uridine triphosphate inhibits both the forward and reverse reactions. All effectors except gibberellic acid are active only in the millimolar range of concentrations; maximal stimulation for any effector is approximately 2-fold. The effects of combinations of effectors are roughly additive. Using pyrophosphate in the presence of magnesium chloride as an effector, results of kinetic studies offer a model by which an effector can activate an enzymatic reaction in one direction and inhibit in the reverse direction.

The Biosynthesis of Sucrose and Nucleoside Diphosphate Glucoses in Phaseolus aureus.

Sucrose-phosphate synthetase is detectable only in intact chloroplast preparations of Phaseolus aureus. In contrast, sucrose synthetase and uridine diphosphate glucose (UDP-glucose) pyrophosphorylase activities are low in extracts of photosynthetic tissues of P. aureus but are high in extracts of nonphotosynthetic tissues. Activities for ADP-, dTDP-, CDP-, and GDP-glucose pyrophosphorylases are generally higher in extracts of photosynthetic tissues of P. aureus than in extracts of nonphotosynthetic tissues. The high levels of sucrose synthetase and of UDP-glucose pyrophosphorylase found in dark-grown hypocotyls begin to decline about 4 hours after exposure to light at a rate of 50% every 3 hours.The data suggest that sucrose-phosphate synthetase and sucrose phosphatase are the enzymes responsible for the biosynthesis of sucrose from photosynthetically fixed CO(2), and that the major function of sucrose synthetase is to catalyze the synthesis of UDP-, ADP-, dTDP-, CDP-, and GDP-glucose from translocated sucrose in nonphotosynthetic tissues; in photosynthetic tissues the pyrophosphorylases may replace sucrose synthetase in catalyzing the synthesis of these nucleoside diphosphate glucoses. We offer the suggestion that sucrose synthetase and UDP-glucose pyrophosphorylase play a major role in the uptake and metabolism of sucrose in nonphotosynthetic tissues. Results are presented from preliminary studies on the conversion in vitro of sucrose to glucose 1-phosphate by the coupled reactions of sucrose synthetase and UDP-glucose pyrophosphorylase with highly purified preparations of these enzymes.

Inhibition of Mung Bean UDP-Glucose: (1-->3)-beta-Glucan Synthase by UDP-Pyridoxal: Evidence for an Active-Site Amino Group.

UDP-pyridoxal competitively inhibits the Ca(2+)-, cellobiose-activated (1-->3)-beta-glucan synthase activity of unfractionated mung bean (Vigna radiata) membranes, with a K(i) of 3.8 +/- 0.7 micromolar, when added simultaneously with the substrate UDP-glucose in brief (3 minute) assays. Preincubation of membranes with UDP-pyridoxal and no UDP-glucose, however, causes progressive reduction of the V(max) of subsequently assayed enzyme and, after equilibrium is reached, 50% inhibition occurs with 0.84 +/- 0.05 micromolar UDP-pyridoxal. This progressive inhibition is reversible provided that the UDP-pyridoxylated membranes are not treated with borohydride, indicating formation of a Schiff's base between the inhibitor and an enzyme amino group. Consistent with this, UDP-pyridoxine is not an inhibitor. The reaction of (1-->3)-beta-glucan synthase with UDP-pyridoxal is stimulated strongly by Ca(2+) and, less effectively, by cellobiose or sucrose, and the enzyme is protected against UDP-pyridoxal by UDP-glucose or by other competitive inhibitors, implying that modification is occurring at the active site. Pyridoxal phosphate is a less potent and less specific inhibitor. Latent (1-->3)-beta-glucan synthase activity inside membrane vesicles can be unmasked and rendered sensitive to UDP-pyridoxal by the addition of digitonin. Treatment of membrane proteins with UDP-[(3)H]pyridoxal and borohydride labels a number of polypeptides but labeling of none of these specifically requires Ca(2+) and sucrose; however, a polypeptide of molecular weight 42,000 is labeled by UDP-[(3)H]pyridoxal in the presence of Mg(2+) and copurifies with (1-->3)-beta-glucan synthase activity.

Gel-Electrophoretic Separation, Detection, and Characterization of Plant and Bacterial UDP-Glucose Glucosyltransferases.

We have developed procedures for detection and characterization of UDP-glucose: glucosyltransferases following electrophoretic separation in nondenaturing polyacrylamide gels. Using digitonin-solubilized membrane protein preparations from a variety of plants and two cellulose-producing bacteria, activity can be demonstrated for several UDP-glucose:beta-glucan synthases with an in situ assay following gel electrophoresis. These enzymes can be characterized within the gels with respect to effector requirements and products produced, and several advantages of this assay over solution assays are demonstrated. For example, the clear dependence of plant UDP-glucose:(1-->3)-beta-glucan synthase on both Ca(2+) and a beta-linked glucoside is shown; bacterial cellulose synthases show direct stimulation within the gel by guanyl oligonucleotide, and the Acetobacter xylinum enzyme appears more stable in the gel assay than in solution assay.

Stimulation of membrane-associated polysaccharide synthetases by a membrane potential in developing cotton fibers.

Conditions which induce a transmembrane electrical potential, positive with respect to the inside of membrane vesicles, result in a substantial (4-12-fold) stimulation of the activity of membrane-associated ß-glucan synthetases in a membrane preparation derived from the developing cotton (Gossypium hirsutum L.) fiber. Induction of electrical potentials which are negative with respect to the inside of the membrane vesicle results in little or no stimulation of ß-glucan synthesis. Those products whose synthesis is stimulated are mainly ß-1,3-glucan, but there is also a considerable increase in ß-1,4-glucan. No α-1,4-glucan (starch) was detected in the reaction products. A transmembrane pH gradient was found to have no effect on ß-glucan synthesis. The results indicate that a transmembrane electrical potential can influence, either directly or indirectly, the activity of membrane-associated polysaccharide synthetases.

Two Kinds of Protein Glycosylation in a Cell-Free Preparation from Developing Cotyledons of Phaseolus vulgaris.

Membrane preparations from developing cotyledons of red kidney bean (Phaseolus vulgaris L.) transferred radioactive mannose from GDP-mannose (U-[(14)C]mannose) to endogenous acceptor proteins. The transfer was inhibited by the antibiotic tunicamycin, suggesting the involvement of lipidoligosaccharide intermediates typical of the pathway for glycosylation of asparagine residues. This was supported by the similarity of the linkage types of radioactive mannose in lipid-oligosaccharide and glycoprotein products; both contained labeled 2-linked mannose, 3,6-linked and terminal mannose typical of glycoprotein "core" oligosaccharides. As expected for "core" glycosylation, the transfer of labeled N-acetylglucosamine (GlcNAc) from UDP-GlcNAc (6-[(3)H]GLcNAc) to 4-linkage in endogenous glycoproteins could also be demonstrated. However, most of the radioactive GlcNAc was incorporated into terminal linkage, in a reaction insensitive to tunicamycin. The proteins receiving "core" oligosaccharide in vitro were heterogeneous in size, in contrast to those receiving most of the GlcNAc (which chiefly comprised the seed reserve-proteins phaseolin and phytohemagglutinin). It is suggested that following "core" glycosylation, single GlcNAc residues are attached terminally to the oligosaccharides of these seed proteins, without the involvement of lipid-linked intermediates. Phaseolin from mature seeds does not possess a significant amount of terminal GlcNAc and so it is possible that these residues are subsequently removed in a processing event.

Protection of cellulose synthesis in detached cotton fibers by polyethylene glycol.

Detachment of the cotton fiber cell from the ovule results in loss of over 90% of the in vivo capacity for synthesis of [(14)C]cellulose from [(14)C]glucose. However, over 50% of the capacity for cellulose synthesis in the detached fiber population is protected when polyethylene glycol 4000 is present during detachment and incubation. Radioautography shows that approximately full capacity is restored in about half the fibers, whereas the other half of the population are incapable of cellulose synthesis from supplied glucose. The rate of cellulose synthesis in such fibers has a pH optimum of 6 and the optimum polyethylene glycol 4000 concentration is 0.06 molal (-9 bars). Cellulose synthesis in such detached fibers is synergistically stimulated by Ca(2+) and Mg(2+) and inhibited by K(+).Evidence is presented which indicates that the protection by polyethylene glycol 4000 is due to its ability to promote membrane resealing, which seems to be required for protecting cellulose synthesis in the detached fiber; however, the requirement for membrane integrity is not exclusively involved in the maintainence of an energy generating system for the synthesis. The possibility that a membrane potential may be required for maintaining an active cellulose synthesizing system is discussed.

Characterization of inhibitors of cellulose synthesis in cotton fibers.

Several compounds were tested for their ability to inhibit the in-vivo synthesis of cellulose and other cell-wall polysaccharides in fibers of cotton (Gossypium hirsutum L.) developing on in-vitro cultured ovules. Inhibitory effects were measured by the ability of the compounds to inhibit the incorporation of radioactivity from [U-(14)C]glucose into these cell-wall polymers. Of the compounds surveyed, 2,6-dichlorobenzonitrile (DCB) was the most effective and specific one for its effects on cellulose synthesis when compared to its effect on the synthesis of other cell-wall components. At 10 µM DCB caused 80% inhibition of cellulose synthesis, and the effect was reversed upon removal of the DCB, with recovery to 90% of the control rate. Two analogs of DCB, 2-chloro-6-fluorobenzonitrile and 2,6-dichlorobenzene carbothiamide, were as specific and nearly as effective as DCB with respect to their effects on cellulose synthesis. Coumarin, generally regarded as an inhibitor of cellulose synthesis in other plant systems, was effective in cotton fibers in millimolar concentrations and, like DCB, was relatively specific with regard to its effect on cellulose synthesis. DCB and coumarin inhibited the synthesis of both primary and secondary wall cellulose. Bacitracin, an inhibitor of the cycling of phosphorylated polyprenols involved in cell-wall synthesis in bacteria, and ethylenediaminetetracetic acid (EDTA) and ethyleneglycol-bis-(ß-amino-ethylether)-N,N'-tetracetic acid (EGTA), chelators of civalent cations, were also effective, although only at relatively high concentrations, in inhibiting incorporation of radioactivity into cellulose.