The Site of Cellulose Synthesis

Shore, Gordon C.; Raymond, Yves; Maclachlan, Gordon

doi:10.1104/pp.56.1.34

Cited by 32 publications

(8 citation statements)

References 23 publications

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“…A moderately high centage (up to 15%) of the High U-CB activity was also recovered in an isolated particulate fraction. This is in contrast to the cell surface glucan synthetase activity described by Shore and MacLachlan in pea stems (17,18); in that case a lower percentage (3-5%) of a similar activity was detected in cell-free homogenates. The initial rates of activity in cotton fibers at early stages of development exceed our calculated rates of in vivo cellulose deposition; however, these rates are obtained using 1 mm UDPPlant Physiol.…”

Section: Resultscontrasting

confidence: 48%

UDP-glucose: Glucan Synthetase in Developing Cotton Fibers

Delmer¹,

Heiniger²,

Kulow³

1977

Plant Physiol.

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In our studies on cellulose biosynthesis in the developing cotton fiber, we have been investigating the utilization of UDPglucose by enzyme systems present in the cotton fiber. We have done this because, for a variety of reasons (3), UDP-glucose can be considered as a likely precursor to cellulose. However, UDPglucose can also serve as a precursor to such compounds as sucrose (4) and steryl glucosides (8, 15) in plants, and it is well known that in a variety of plant tissues, UDP-glucose can also serve as a substrate for the synthesis of /-,(1 -* 3)-glucan (2, 5, 6, 20-22), or for the synthesis of a glucan with mixed f3-(1 -+ 3) and 18-(1 --4) linkages (14, 19).We have studied the utilization of UDP-glucose by cotton fibers harvested at the onset of secondary wall cellulose synthesis, determining and comparing the activities both in fibers detached from the ovules ("semi-intact fibers"), and in a particulate fraction isolated from homogenized fibers. Since UDPglucose can serve as substrate for several different enzyme systems, we have analyzed for a variety of possible reaction prod- (224 ,uCi/,umol) was purchased from New England Nuclear. f8-(1--+3)-Glucanase purified from Rhizopus was the generous gift of E. T. Reese, U.S. Army Laboratories, Natick, Mass. Laminaribiose was prepared by digestion of laminarin (Sigma) with this 63(1-.3)-glucanase.The disaccharide was purified from the digestion mixture by chromatography on a Bio-Gel P-2 column.Determination of Radioactivity. Samples were dried on paper or filters and counted in toluene scintillation fluid (2.2 g PPO and 176 mg POPOP/l toluene). Aqueous samples, samples in chloroform-methanol, or undried fibers were counted in the same toluene scintillation fluid to which '/lo volume of Bio-Solve (Beckman) has been added. All samples were counted in a Packard Tri-Carb model 3390 liquid scintillation spectrometer. Quench curves for various counting conditions were constructed, and where appropriate, corrections to dpm were made.Detached Fiber Assay. Fibers of known age were removed from the boll, divided into 50-mg aliquots, and placed in tubes containing 2 ml 0.01 M TES buffer (pH 7.5) containing 1 mm EDTA. The fibers were agitated on a Vortex mixer with a small spatula in the tube until thoroughly wetted (about 30 sec). The individual aliquots were then removed from the tubes, drained on paper towels, and a single aliquot was placed into each reaction mixture at room temperature to start the enzyme assay.

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Section: Resultscontrasting

confidence: 48%

UDP-glucose: Glucan Synthetase in Developing Cotton Fibers

Delmer¹,

Heiniger²,

Kulow³

1977

Plant Physiol.

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“…5 shows the distribution of cellulase activity in the particulate fraction after isopycnic centrifugation in linear sucrose gradients. In the absence of added MgCls, ribosomes dissociate from the ER, and all ER and marker enzymes are found in a single locus at a density of approximately 1.11 (19). Cellulase activity is found in this region of the gradient.…”

Section: Localization Of Cehulases In Subcellular Fractionsmentioning

confidence: 93%

“…Apical segments (10 mm) of dark-grown pea epicotyls were harvested at zero time or 2 days after 2,4-D treatment and chopped with razor blades (13,19) in 0.3 vol of extraction medium containing 0.4 M sucrose (ribonuclease free), 100 mM Tris-HCl (pH 8.0 at 2~ and 5 mM dithiothreitol with or without MgCI2. The slurry was squeezed through nylon cloth, and the filtrate was centrifuged at 500 g for 20 min.…”

Section: Fractionation Of Subceilular Componentsmentioning

confidence: 99%

Subcellular localization of cellulases in auxin-treated pea.

Bal¹,

Verma²,

Byrne³

et al. 1976

The Journal of Cell Biology

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Two forms of cellulase, buffer soluble (BS) and buffer insoluble (BI), are induced as a result of auxin treatment of dark-grown pea epicotyls. These two cellulases have been purified to homogeneity. Antibodies raised against the purified cellulases were conjugated with ferritin and were used to localize the two cellulases. Tissue sections were fixed in cold paraformaldehyde-glutaraldehyde and incubated for 1 h in the ferritin conjugates. The sections were washed with continuous shaking for 18 h and subsequently postfixed in osmium tetroxide. Tissue incubated in unconjugated ferritin was used as a control. A major part of BI cellulase is localized at the inner surface of the cell wall in close association with microfibrils. BS cellulase is localized mainly within the distended endoplasmic reticulum. Golgi complex and plasma membrane appear to be completely devoid of any cellulase activity. These observations are consistent with cytochemical localization and biochemical data on the distribution of these two cellulases among various cell and membrane fractions.Cellulolytic enzymes (~-l,4-glucan, 4-glucanohydrolase, EC 3.2.1.4) occur in specific locations in tissues of higher plants at particular stages of development. Their possible regulatory roles in cell growth and differentiation (1, 7-10, 12, 14-18), and the hormonal control of their induction (5-8, 21), have been emphasized in earlier work. Although cellulases have been purified and partly characterized (5, 6), their actual localization at the cellular level has not been demonstrated. In elucidating the physiological role of these enzymes, it is imperative that their exact subcellular loci be defined. Attempts made to localize cellulase in higher plants (3, 4) and snail (20) have not met the refinements necessary for precise ultrastructural localization.Auxin-induced growth in etiolated pea epicotyls offers a unique system for localization of cellulases. Two different species of cellulases, one buffer insoluble (Bi) and the other buffer soluble (BS), have been purified and characterized from this system (5, 6). These two proteins have proved to be immunologically different. With the use of ferritin-conjugated antibodies to these two cellulases, an immunocytochemical method was developed to localize the enzymes at subcellular levels. The results were supplemented by cytochemical (4) and cell fractionation procedures. MATERIALS AND METHODS Plant Material and Hormone TreatmentSeeds of Pisum sativum L. var. Alaska were soaked for 20 min in 0.5% sodium hypochlorite. After 8 h of imbibition in tap water, seeds were planted in moistened vermiculite and grown in darkness at room temperature.

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“…prompted by reports of Shore and Maclachlan (37,38) that a "cell surface cellulose synthetase system" could be detected in pea tissue by supplying radioactive UDPG to tissue slices. They presented no evidence for the nature of the radioactive product or for its location within the cells of the treated tissue.…”

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confidence: 99%

Labeling of the Plasma Membrane of Pea Cells by a Surface-localized Glucan Synthetase

Anderson¹,

Ray²

1978

Plant Physiol.

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When radioactive UDP-glucose is supplied to 1-millimeter-thick slices of pea (Pisum satidum) stem tissue, radioactive glucose becomes incorporated into membrane-bound polysaccharides. Evidence is given that this incorporation does not result from breakdown of UDP-glucose and utilization of the resultant free glucose, and that the incorporation most likely takes place at the ceUl surface, leading to a specific labeling of the plasma membrane. The properties of the plasma membrane that are indicated by this method of recognition, including the association of K+-stimulated ATPase activity with the plasma membrane, resemble properties inferred using other approaches. The membrane-associated polysaccharide product formed from UDP-glucose is largely 1,3-linked glucan, presumably cailose, and does not behave as a precursor of cel wall polymers. No substantial amount of cellulose is formed from UDP-glucose in this procedure, even though these cells incorporate free glucose rapidly Into ceUlulose. This synthetase system that uses external UDP-glucose may serve for formation of wound callose.Development of procedures for isolation of the plasma membrane of plant cells has been retarded by uncertainties about how plasma membrane fragments may be recognized and distinguished from other cellular membranes. The technique that has been used in the majority of investigations, staining of thin sections with phosphotungstic-chromic acid (34), is laborious, difficult to quantify, and is found by some workers to stain membranes other than the plasma membrane (10,26,39,44). Monovalent salt-stimulated ATPase has been emphasized as a probable marker enzyme for the plasma membrane (17, 18) but this type of activity may also occur in the tonoplast and other intracellular membranes (16,22,36). Glucan synthetase assayed using high concentrations of UDPG2 has also been proposed as a plasma membrane marker (12,17,28,39,43) but glucan synthetase activity is also found in Golgi membranes (30,43) and no evidence has been presented for an assay of this type that is quantitatively specific for plasma membranes.The approach that has most often been used for recognizing plasma membranes derived from animal cells, that of labeling the external surface of intact cells with a nonpenetrating reactive reagent prior to cell disruption, has proved unsuccessful in a number of attempts with plant material (11,25), but some recent reports infer success in specifically labeling the plasma membrane in tissues (15, 46) or using protoplasts (1,8). This report presents evidence for a different method of specifically labeling the plasma membrane, by making use of a glucan synthetase activity that utilizes UDPG supplied outside the cell. These studies were ' Supported by a grant to P. M. R. from the National Science Foundation.2Abbreviations: UDPG, uridine diphospho-D-glucose.prompted by reports of Shore and Maclachlan (37, 38) that a "cell surface cellulose synthetase system" could be detected in pea tissue by supplying radioactive UDPG to tissue slices....

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The Site of Cellulose Synthesis

Abstract: ABSTRACT,B-1,4-Glucan (cellulose) synthetase activity (UDP-glucose:,3-1 ,4-glucan-glucosyl transferase) present at cell surfaces of growing regions of Pisum sativum epicotyl was assayed by supplying UDP-'4C-glucose directly to thin slices of tissue.

Cited by 32 publications

References 23 publications

UDP-glucose: Glucan Synthetase in Developing Cotton Fibers

UDP-glucose: Glucan Synthetase in Developing Cotton Fibers

Subcellular localization of cellulases in auxin-treated pea.

Labeling of the Plasma Membrane of Pea Cells by a Surface-localized Glucan Synthetase

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