3D volumes constructed from pixel-based images by digitally clearing plant and animal tissue.

Construction of three-dimensional volumes from a series of two-dimensional images has been restricted by the limited capacity to decrease the opacity of tissue. The use of commercial software that allows colour-keying and manipulation of two-dimensional images in true three-dimensional space allowed us to construct three-dimensional volumes from pixel-based images of stained plant and animal tissue without generating vector information. We present three-dimensional volumes of (1) the crown of an oat plant showing internal responses to a freezing treatment, (2) a sample of a hepatocellular carcinoma from a woodchuck liver that had been heat-treated with computer-guided radiofrequency ablation to induce necrosis in the central portion of the tumour, and (3) several features of a sample of mouse lung. The technique is well suited to images from large sections (greater than 1 mm) generated from paraffin-embedded tissues. It is widely applicable, having potential to recover three-dimensional information at virtually any resolution inherent in images generated by light microscopy, computer tomography, magnetic resonance imaging or electron microscopy.

Roles of microtubules and cellulose microfibril assembly in the localization of secondary-cell-wall deposition in developing tracheary elements.

The roles of cellulose microfibrils and cortical microtubules in establishing and maintaining the pattern of secondary-cell-wall deposition in tracheary elements were investigated with direct dyes to inhibit cellulose microfibril assembly and amiprophosmethyl to inhibit microtubule polymerization. When direct dyes were added to xylogenic cultures of Zinnia elegans L. mesophyll cells just before the onset of differentiation, the secondary cell wall was initially secreted as bands composed of discrete masses of stained material, consistent with immobilized sites of cellulose synthesis. The masses coalesced, forming truncated, sinuous or smeared thickenings, as secondary cell wall deposition continued. The absence of ordered cellulose microfibrils was confirmed by polarization microscopy and a lack of fluorescence dichroism as determined by laser scanning microscopy. Indirect immunofluorescence showed that cortical microtubules initially subtended the masses of dye-altered secondary cell wall material but soon became disorganized and disappeared. Although most of the secondary cell wall was deposited in the absence of subtending cortical microtubules in dye-treated cells, secretion remained confined to discrete regions of the plasma membrane. Examination of non-dye-treated cultures following application of microtubule inhibitors during various stages of secondary-cell-wall deposition revealed that the pattern became fixed at an early stage such that deposition remained localized in the absence of cortical microtubules. These observations indicate that cortical microtubules are required to establish, but not to maintain, patterned secondary-cell-wall deposition. Furthermore, cellulose microfibrils play a role in maintaining microtubule arrays and the integrity of the secondary-cell-wall bands during deposition.

Sucrose phosphate synthase activity rises in correlation with high-rate cellulose synthesis in three heterotrophic systems.

Based on work with cotton fibers, a particulate form of sucrose (Suc) synthase was proposed to support secondary wall cellulose synthesis by degrading Suc to fructose and UDP-glucose. The model proposed that UDP-glucose was then channeled to cellulose synthase in the plasma membrane, and it implies that Suc availability in cellulose sink cells would affect the rate of cellulose synthesis. Therefore, if cellulose sink cells could synthesize Suc and/or had the capacity to recycle the fructose released by Suc synthase back to Suc, cellulose synthesis might be supported. The capacity of cellulose sink cells to synthesize Suc was tested by analyzing the Suc phosphate synthase (SPS) activity of three heterotrophic systems with cellulose-rich secondary walls. SPS is a primary regulator of the Suc synthesis rate in leaves and some Suc-storing, heterotrophic organs, but its activity has not been previously correlated with cellulose synthesis. Two systems analyzed, cultured mesophyll cells of Zinnia elegans L. var. Envy and etiolated hypocotyls of kidney beans (Phaseolus vulgaris), contained differentiating tracheary elements. Cotton (Gossypium hirsutum L. cv Acala SJ-1) fibers were also analyzed during primary and secondary wall synthesis. SPS activity rose in all three systems during periods of maximum cellulose deposition within secondary walls. The Z. elegans culture system was manipulated to establish a tight linkage between the timing of tracheary element differentiation and rising SPS activity and to show that SPS activity did not depend on the availability of starch for degradation. The significance of these findings in regard to directing metabolic flux toward cellulose will be discussed.

Carbon partitioning to cellulose synthesis.

This article discusses the importance and implications of regulating carbon partitioning to cellulose synthesis, the characteristics of cells that serve as major sinks for cellulose deposition, and enzymes that participate in the conversion of supplied carbon to cellulose. Cotton fibers, which deposit almost pure cellulose into their secondary cell walls, are referred to as a primary model system. For sucrose synthase, we discuss its proposed role in channeling UDP-Glc to cellulose synthase during secondary wall deposition, its gene family, its manipulation in transgenic plants, and mechanisms that may regulate its association with sites of polysaccharide synthesis. For cellulose synthase, we discuss the organization of the gene family and how protein diversity could relate to control of carbon partitioning to cellulose synthesis. Other enzymes emphasized include UDP-Glc pyrophosphorylase and sucrose phosphate synthase. New data are included on phosphorylation of cotton fiber sucrose synthase, possible regulation by Ca2+ of sucrose synthase localization, electron microscopic immunolocalization of sucrose synthase in cotton fibers, and phylogenetic relationships between cellulose synthase proteins, including three new ones identified in differentiating tracheary elements of Zinnia elegans. We develop a model for metabolism related to cellulose synthesis that implicates the changing intracellular localization of sucrose synthase as a molecular switch between survival metabolism and growth and/or differentiation processes involving cellulose synthesis.

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.

A Secreted Factor Inducs Cell Expansion and Formation of Metaxylem-Like Tracheary Elements in Xylogenic Suspension Cultures of Zinnia.

Conditioned medium from mesophyll cell-suspension cultures of Zinnia elegans L. has striking effects on cell expansion and tracheary element differentiation when applied to cultures of freshly isolated mesophyll cells. These effects include (a) induction of early cell expansion, (b) delay in differentiation by 48 h or more, (c) reduction in the synchrony of differentiation, and (d) early formation of very large, metaxylem-like tracheary elements. Like reduced osmotic potential and buffering at pH 5.5, conditioned medium appears to have its primary effect on cell expansion. Partial characterization of the expansion-inducing factor indicates that it is heat stable, of low molecular mass, and is resistant to protease. It also binds reversibly to concanavalin A but is not adsorbed by charcoal. We suggest that the secreted factor may be an oligosaccharide involved in the coordination of cell expansion and differentiation and the regulation of the protoxylem-like to metaxylem-like transition in xylogenic suspension cultures.

Cellulose microfibrils, cell motility, and plasma membrane protein organization change in parallel during culmination in Dictyostelium discoideum.

Prestalk cells of Dictyostelium discoideum contribute cellulose to two distinct structures, the stalk tube and the stalk cell wall, during culmination. This paper demonstrates by freeze fracture electron microscopy that two distinct types of intramembrane particle aggregates, which can be characterized as cellulose microfibril terminal complexes, occur in the plasma membranes of cells synthesizing these different forms of cellulose. The same terminal complexes were observed in situ in developing culminants and in vitro in monolayer cells induced to synthesize the two types of cellulose. We propose that cessation of cell motility is associated with a change in packing and intramembrane mobility of the particle aggregates, which cause a change in the nature of the cellulose synthesized. The terminal complexes are compared to those described in other organisms and related to the previous hypothesis of two modes of cellulose synthesis in Dictyostelium.

Detection of cellulose with improved specificity using laser-based instruments.

Specific detection of cellulose has not been possible using laser based instruments such as laser scanning confocal microscopes (LSCM) and fluorescently activated cell sorters (FACS). Common cellulose dyes are nonspecific and/or nonexcitable with common lasers. Furthermore, many lasers emit wavelengths that overlap with autofluorescence from chlorophyll and other plant molecules. We demonstrate that a cellulase and an isolated bacterial cellulose binding domain (CBD) conjugated to fluorescent dyes can be used for laser detection of cellulose with improved specificity. Cell walls of differentiating tracheary elements and spores of Dictyostelium discoideum were tested in this study. For double labeling, autofluorescence interfering with the rhodamine signal was eliminated by collecting each excitation channel separately followed by computer recombination or by using a narrow band pass barrier filter allowing simultaneous channel collection. Using these methods, cellulose and microtubules tagged with a monoclonal antibody to alpha-tubulin were effectively colocalized in chlorophyll-containing tracheary elements using a LSCM. Also, Dictyostelium discoideum spores labeled or unlabeled with CBD-FITC were separated into two populations by FACS indicating that this tag should be useful in future mutagenesis experiments. Therefore, the presence or absence of cellulose can now be analyzed using common lasers.

A membrane-associated form of sucrose synthase and its potential role in synthesis of cellulose and callose in plants.

Sucrose synthase (SuSy; EC 2.4.1.13; sucrose + UDP reversible UDPglucose + fructose) has always been studied as a cytoplasmic enzyme in plant cells where it serves to degrade sucrose and provide carbon for respiration and synthesis of cell wall polysaccharides and starch. We report here that at least half of the total SuSy of developing cotton fibers (Gossypium hirsutum) is tightly associated with the plasma membrane. Therefore, this form of SuSy might serve to channel carbon directly from sucrose to cellulose and/or callose synthases in the plasma membrane. By using detached and permeabilized cotton fibers, we show that carbon from sucrose can be converted at high rates to both cellulose and callose. Synthesis of cellulose or callose is favored by addition of EGTA or calcium and cellobiose, respectively. These findings contrast with the traditional observation that when UDPglucose is used as substrate in vitro, callose is the major product synthesized. Immunolocalization studies show that SuSy can be localized at the fiber surface in patterns consistent with the deposition of cellulose or callose. Thus, these results support a model in which SuSy exists in a complex with the beta-glucan synthases and serves to channel carbon from sucrose to glucan.

Cell Expansion and Tracheary Element Differentiation Are Regulated by Extracellular pH in Mesophyll Cultures of Zinnia elegans L.

The effects of medium pH on cell expansion and tracheary element (TE) differentiation were investigated in differentiating mesophyll suspension cultures of Zinnia elegans L. In unbuffered cultures initially adjusted to pH 5.5, the medium pH fluctuated reproducibly, decreasing about 1 unit prior to the onset of TE differentiation and then increasing when the initiation of new Tes was complete. Elimination of large pH fluctuations by buffering the culture medium with 20 mM 2-(N-morpholino)ethanesulfonic acid altered both cell expansion and TE differentiation, whereas altering the starting pH of unbuffered culture medium had no effect on either process. Cell expansion in buffered cultures was pH dependent with an optimum of 5.5 to 6.0. The direction of cell expansion was also pH dependent in buffered cultures. Cells elongated at pH 5.5 to 6.0, whereas isodiametric cell expansion was predominant at pH 6.5 to 7.0. The onset of TE differentiation was delayed when the pH was buffered higher or lower than 5.0. However, TEs eventually appeared in cultures buffered at pH 6.5 to 7.0, indicating that a decrease in pH to 5.0 is not necessary for differentiation. Very large TEs with secondary cell wall thickenings resembling metaxylem differentiated in cultures buffered at pH 5.5 to 6.0, which also showed the greatest cell expansion. The correlation between cell expansion and delayed differentiation of large, metaxylem-like TEs may indicate a link between the regulatory mechanisms controlling cell expansion and TE differentiation.

Effects of cycling temperatures on fiber metabolism in cultured cotton ovules.

The effects of temperature on rates of cellulose synthesis, respiration, and long-term glucose uptake were investigated using cultured cotton ovules (Gossypium hirsutum L. cv Acala SJ1). Ovules were cultured either at constant 34 degrees C or under cycling temperatures (12 h at 34 degrees C/12 h at 15-40 degrees C). Rates of respiration and cellulose synthesis at various temperatures were determined on day 21 during the stage of secondary wall synthesis by feeding cultured ovules with [(14)C]glucose. Respiration increased between 18 and approximately 34 degrees C, then remained constant up to 40 degrees C. In contrast, the rate of cellulose synthesis increased above 18 degrees C, reached a plateau between about 28 and 37 degrees C, and then decreased at 40 degrees C. Therefore, the optimum temperature for rapid and metabolically efficient cellulose synthesis in Acala SJ1 is near 28 degrees C. In ovules cycled to 15 degrees C, respiration recovered to the control rate immediately upon rewarming to 34 degrees C, but the rate of cellulose synthesis did not fully recover for several hours. These data indicate that cellulose synthesis and respiration respond differently to cool temperatures. The long-term uptake of glucose, which is the carbon source in the culture medium, increased as the low temperature in the cycle increased between 15 and 28 degrees C. However, glucose uptake did not increase in cultures grown constantly at 34 degrees C compared to those cycled at 34/28 degrees C. These observations are consistent with previous observations on the responses of fiber elongation and weight gain to cycling temperatures in vitro and in the field.

Cultured Ovules as Models for Cotton Fiber Development under Low Temperatures.

Cotton fibers (Gossypium hirsutum L.) developing in vitro responded to cyclic temperature change similarly to those of field-grown plants under diumal temperature fluctuations. Absolute temperatures and rates of temperature change were similar under both conditions. In vitro fibers exhibited a "growth ring" for each time the temperature cycled to 22 or 15 degrees C. Rings were rarely detected when the low point was 28 degrees C. The rings seemed to correspond to alternating regions of high and low cellulose accumulation. Fibers developed in vitro under 34 degrees C/22 degrees C cycling developed similarly to constant 34 degrees C controls, but 34 degrees C/22 degrees C and 34 degrees C/15 degrees C cycling caused delayed onset and prolonged periods of elongation and secondary wall thickening. Control fiber length and weight were finally achieved under 34 degrees C/22 degrees C cycling, but both parameters were reduced at the end of the experiment under 34 degrees C/15 degrees C cycling. Fibers developed under all conditions had equal bundle tensile strength. These results demonstrate that: (a) cool temperature effects on fiber development are at least partly fiber/ovule-specific events; they do not depend on whole-plant physiology; and (b) cultured ovules are valid models for research on the regulation of the field cool temperature response.

Tracheary-element differentiation in suspension-cultured cells ofZinnia requires uptake of extracellular Ca(2+) : Experiments with calcium-channel blockers and calmodulin inhibitors.

Tracheary-element (TE) differentiation in suspension cultures ofZinnia elegans L. mesophyll cells was inhibited by blocking calcium uptake in three ways: 1) reducing the [Ca(2+)] of the culture medium, 2) blocking calcium channels with the non-permeant cation La(3+), and 3) blocking calcium channels with permeant dihydropyridine calcium-channel blockers. Calcium-channel blockers were effective when added at any time between 0 and 48 h after culture initiation; after 48h, calcium sequestration and secondary cell-wall deposition began. In contrast, calmodulin antagonists inhibited TE differentiation when added at the beginning of culture, but not when added after 24h. These results indicate that TE differentiation involves at least two calcium-regulated events: one calmodulin-dependent and occurring shortly after exposure to inductive conditions, and the other calmodulin-independent and occurring just prior to secondary cell-wall deposition.

Abscisic Acid Control of Lectin Accumulation in Wheat Seedlings and Callus Cultures : Effects of Exogenous ABA and Fluridone.

Wheat germ agglutinin is found in wheat embryos and a similar lectin is present in the roots of older plants. We report here that 10 micromolar abscisic acid (ABA) produces an average two to three-fold enhancement in the amount of lectin in the shoot base and the terminal portion of the root system of hydroponically grown wheat seedlings. Although ABA stunts seedling growth, a similar growth inhibition produced by ancymidol is not accompanied by elevated lectin levels. To further clarify the role of ABA, wheat callus cultures were employed. Callus derived from immature embryos was grown on growth medium containing various combinations of ABA and 2,4-dichlorophenoxyacetic acid. Those grown in the presence of 10 micromolar ABA exhibit the largest increases in lectin compared to material grown on other regimes. The involvement of ABA in lectin accumulation was further probed with fluridone, an inhibitor of carotenoid synthesis which has also been linked to depressed levels of endogenous ABA. Wheat seedlings grown in the presence of 1 or 10 milligrams per liter fluridone have few or no carotenoids, and wheat germ agglutinin levels in the shoot base and roots are lower compared to controls. The greatest effect (a 39% reduction in the shoot base) is produced at an herbicide concentration of 10 milligrams per liter. Exogenous 10 micromolar ABA greatly stimulates lectin accumulation in the presence of fluridone, but the levels are not as high as those produced by ABA alone. These results indicate that lectin synthesis is under ABA control in both wheat embryos and adult plants.

Alteration of in vivo cellulose ribbon assembly by carboxymethylcellulose and other cellulose derivatives.

In vivo cellulose ribbon assembly by the Gram-negative bacterium Acetobacter xylinum can be altered by incubation in carboxymethylcellulose (CMC), a negatively charged water-soluble cellulose derivative, and also by incubation in a variety of neutral, water-soluble cellulose derivatives. In the presence of all of these substituted celluloses, normal fasciation of microfibril bundles to form the typical twisting ribbon is prevented. Alteration of ribbon assembly is most extensive in the presence of CMC, which often induces synthesis of separate, intertwining bundles of microfibrils. Freeze-etch preparations of the bacterial outer membrane suggest that particles that are thought to be associated with cellulose synthesis or extrusion may be specifically organized to mediate synthesis of microfibril bundles. These data support the previous hypothesis that the cellulose ribbon of A. xylinum is formed by a hierarchical, cell-directed, self-assembly process. The relationship of these results to the regulation of cellulose microfibril size and wall extensibility in plant cell walls is discussed.

Cellulose biogenesis: Polymerization and crystallization are coupled processes in Acetobacter xylinum.

Calcofluor White ST, stilbene derivative used commerically as an optical brightener for cellulose, increased the rate of glucose polymerization into cellulose by resting cells of the gram-negative bacterium Acetobacter xylinum. This bacterium normally produces a ribbon of cellulose that is a composite of crystalline microfibrils. In concentrations above 0.1 mM, Calcofluor disrupts the assembly of crystalline cellulose I microfibrils and their integration into a composite ribbon by stoichiometric binding to glucose residues of newly polymerized glucan chains. Under these conditions, the rate of glucose polymerization increases up to 4 times the control rate, whereas oxygen uptake increases only 10-15%. These observed effects are readily reversible. If free Calcofluor is washed away or depleted below the threshold value by binding to cellulose as polymerization continues, ribbon production and the normal rate of polymerization resume. It is concluded that polymerization and crystallization are cell-directed, coupled processes and that the rate of crystallization determines the rate of polymerization. It is suggested that coupling must be maintained for biogenesis of crystalline cellulose I.

Calcofluor white ST Alters the in vivo assembly of cellulose microfibrils.

The fluorescent brightener, Calcofluor White ST, prevents the in vivo assembly of crystalline cellulose microfibrils and ribbons by Acetobacter xylinum. In the presence of more than 0.01 percent Calcofluor, Acetobacter continues to synthesize high-molecular-weight beta-1,4 glucans. X-ray crystallography shows that the altered product exhibits no detectable crystallinity in the wet state, but upon drying it changes into crystalline cellulose I. Calcofluor alters cellulose crystallization by hydrogen bonding with glucan chains. Synthesis of this altered product is reversible and can be monitored with fluorescence and electron microscopy. Use of Calcofluor has made it possible to separate the processes of polymerization and crystallization leading to the biogenesis of cellulose microfibrils, and has suggested that crystallization occurs by a cell-directed. self-assembly process in Acetobacter xylinum.