New approaches to the study of cellulose biosynthesis

Abstract

Examples of a variety of approaches for studying the mechanism and regulation of cellulose biosynthesis are presented. Attempts to demonstrate conclusively a cellulose synthase activity using membrane preparations derived from higher plants have not been successful; the predominant UDP-glucose: /3-glucan-/3-glucosyltransferase detected in these preparations is a /3-(l->3)-glucan synthase that is dependent upon Ca2+ and a /3-glucobiose, such as laminaribiose or cellobiose, for activity. Xyloglucan glucosyltransferase activity is detected in all plant preparations examined and, in cotton fibres, the activity of this enzyme correlates well with the level of xyloglucan found in the fibre. Low activity for a Mg2+-dependent /3-(l-»4)-glucan synthase is found in extracts from soy beans and mung beans, but not cotton fibres; this enzyme could represent either a dissociated form of xyloglucan glucosyltransferase or a partially latent form of cellulose synthase. In vitro translation of RNA from developing cotton fibres shows notable increases in the level of several relatively abundant translatable mRNAs associated with the time of onset of secondary-wall cellulose synthesis. In order to determine whether these apparent changes in gene expression represent enhanced expression of specific genes required for cellulose synthesis, several strategies are being developed for identification of specific polypeptides required for this process. One strategy involves the successful development of a technique for detection of glucan synthase activity in acrylamide gels, a technique that should prove useful for characterization of the polypeptide composition of such enzymes. We have also synthesized a photoaffinity analogue of 2,6-dichlorobenzonitrile (DCB), a potent and specific inhibitor of cellulose synthesis. The analogue is also an effective inhibitor in vivo, and upon ultraviolet irradiation of extracts in the presence of the radioactive analogue, we observe a relatively specific labelling of a polypeptide of 18 X 103 Mr. Finally, we have studied the spatial regulation and structural requirements for cellulose synthesis in internode cells of the alga Chara corallina. Cellulose deposition in vivo shows spatial localization, which correlates with acid and base bands along the cell. Using internodes perfused with solutions containing UDP-[14C]glucose and subsequently ligated, we were able to demonstrate synthesis of a highly insoluble cell-wall-localized glucan, thus offering hope that Chara can be developed as another useful system for studying the mechanism and regulation of cellulose synthesis. I N T R O D U C T I O NSince microfibrillar cellulose is a major structural component of the cell walls of higher plants, and many algae and fungi, regulation of the extent and pattern of deposition of this polymer must certainly be involved in the overall regulation of growth and development in these organisms. Thus, it is regrettable that biochemical studies on the mechanism of synthesis of cellulose have proven so difficult; our lack o...

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

“…It has been speculated that the same enzyme system in higher plants may form both/3-1,3-and/3-1,4-linkages, and that cell disruption (whether from a wound in nature or an isolation procedure in the laboratory) causes a conformational change in the synthetic or regulatory proteins such that /3-1,3-1inked glucans are synthesized rather than cellulose (Jacob and Northcote 1985;Delmer et al 1985;Delmer 1987;Northcote 1989). The formation of 1,3-linkages by the D. discoideum membrane preparations and their absence from the native stalks indicates that there may be similar regulation of glucan synthesis in the cellular slime molds and higher plants.…”

“…Membrane preparations from higher-plant cells incorporate large amounts of glucose from uridine 5'-diphosphate glucose (UDPGlc) into a glucose polymer, but this invariably turns out to be predominantly/%l,3-1inked rather than /%l,4-1inked. Various hypotheses have been advanced to explain this phenomenon: perhaps the callose and cellulose-synthase systems are the same and cell disruption activates callose synthesis preferentially (Jacob and Northcote 1985;Delmer et al 1985;Delmer 1987); or perhaps a regulatory protein as part of the cellulosesynthase complex is rapidly degraded upon cell disruption (Delmer 1988).…”

In situ synthesis of β-glucan microfibrils on tobacco plasma membrane sheets

“…Methylation of ␤-glucan products was performed by the method of Harris et al (13). The methylated derivatives were separated by TLC on silica gel at 30°C for 120 min by using 1-hexane͞diethyl ether͞acetic acid (6:3:1, vol͞vol) as solvent (14). T-Glc, 3-Glc, and 4-Glc indicate the derivatives obtained from terminal, 3-linked and 4-linked glucosyl residues, respectively.…”

The cellulose synthase gene of Dictyostelium

“…Yet, Dictyostelium accumulates no measurable callose in vivo. Membrane preparations from plants synthesize in vitro almost exclusively callose (produced abundantly as a wound response in vivo) in preference to cellulose (32), perhaps because the same enzyme is responsible for the synthesis of both and that cell disruption results in a change in the linkage formed (33,34). This hypothesis is supported by our observation that membrane preparations from dcsA Ϫ cells synthesize neither (134)-␤-nor (133)-␤-linked glucans nor any glucan polymer for that matter.…”