In vitro B-glucan products were synthesized by digitonin-solubilized enzyme preparations from plasma membrane-enriched fractions of cotton (Cossypium hirsutum) fiber cells. The reaction mixture favoring &1,4-glucan synthesis included the following effedors: MgZ+, Caz+, cellobiose, cyclic-3':5'-CMP, and digitonin. The ethanol insoluble fraction from this reaction contained &1,4-glucan and 8-1,3-glucan in an approximate ratio of 25:69. Approximately 16% of the &1,4-glucan was resistant to the acetic/nitric acid reagent. The x-ray diffraction pattern of the treated produd favoring &l,4-glucan synthesis strongly resembled that of cellulose II. O n the basis of methylation analysis, the acetic/nitric acid reagent-insoluble glucan produd was found to be exclusively 8-1,4-linked. Enzymic hydrolysis confirmed that the produd was hydrolyzed only by cellobiohydrolase 1. Autoradiography proved that the produd was synthesized in vitro. l h e degree of polymerization (DP) of the in vitro product was estimated by nitration and size exclusion chromatography; there were two average DPs of 59 (70%) and 396 (30%) for the @-1,3-glucanase-treated sample, and an average DP of 141 for the acetic/nitric acid reagent-insoluble produd. O n the basis of product analysis, the positive identification of in vitro-synthesized cellulose was established.Cellulose is the most abundant macromolecule on earth (Brown, 1985). It serves a major structural role in the cell wall of plants, some algae, and certain fungi and is the primary component of economical products such as wood, cotton, and paper. Because of its tremendous abundance and its physiological and economical importance, many attempts at in vitro cellulose synthesis have been made with cell-free systems from various sources during the past three decades Brown, 1989b;Read and Delmer, 1991). The greatest progress has been made using Acetobacter xylinum as an experimental model (Ross et al., 1987;Brown, 1989a;Lin and Brown, 1989;Lin et al., 1990;Saxena et al., 1990;Wong et al., 1990;Mayer et al., 1991;Saxena et al., 1991). In higher plants, however, numerous failures to detect activity for in vitro cellulose synthase have continued because it has remained impossible to make preparations from higher plant cells capable of synthesizing true microfibrillar cellulose or even appreciable quantities of (3-1,4-glucan (Read and Delmer, 1991).Severa1 methods for the characterization of products synthesized in vitro, such as solubility properties, acid hydrolysis, acetolysis, specific enzymic digestion, and linkage analysis after periodate oxidation or methylation, have been used either alone or in various combinations (Fry, 1988). Insolubility in strong alkali (eg. 24% KOH) has been used for the detection of p-1,4-glucan, but (3-1,3-glucan synthesized in vitro may also be present in the alkali-insoluble fraction (Hayashi et al., 1987). The AN reagent (Updegraff, 1969) has been used for the selection of cellulose because only crystalline cellulose can remain after treatment with this reagent. The mo...

In vitro assembly of cellulose from plasma membrane extracts of the cotton (Gossypium hirsutum) fiber was enriched by a combination of 3 4 N-morpholino)propanesulfonic acid extraction buffer and two independent digitonin solubilization steps consisting of 0.05% digitonin (SE,) followed by 1 % digitonin (SE,). Clucan synthase activity assays revealed that, although the SE, fraction possessed higher activity, only 8.6% of the in vitro product survived aceticl nitric acid treatment. On the other hand, the SE, fraction was less active, but 32.1 % of the total glucan in vitro product was resistant to acetichitric acid. In vitro products synthesized from the SE, fraction contained P-1,3-glucan and fibrillar cellulose I, whereas the SE, fraction produced P-1,3-glucan and cellulose II. Both celluloses assembled in vitro were labeled with cellobiohydrolase Igold complex, and the electron diffraction patterns of both products from SE, and SE, revealed cellulose I and cellulose II, respectively. Contamination of native cellulose was ruled out by extensive evidente from autoradiography of the ethanol-insoluble and aceticl nitric acid-insoluble materials, including three different controls.To understand the mechanism of cellulose biosynthesis, many attempts to synthesize cellulose in vitro have been made with cell-free systems from different sources (Franz and Heininger, 1981;Robinson and Quader, 1981;Carpita, 1982;Maclachan, 1982;Blaschek et al., 1983;Delmer, 1987;Brown, 1989aBrown, , 1989bDhugga and Ray, 1991;Read and Delmer, 1991). The greatest progress has been made using Acetobacter xylinum as an experimental model system, and sufficiently high rates of synthesis of P-1,4-glucan from UDP-Glc were achieved (Aloni et al

The regulation and kinetic properties of cellulose synthase as well as j3-1,3-glucan synthase have been studied. The cellulose was detected using acetic/nitric acid insolubility as an indicator of cellulose (this product contained only j3-1,4-linked glucans; K. Okuda, L. Li, K. Kudlicka, S. Kuga Previous enzyme assay methods for cellulose synthase have been based on alkali-insoluble products; however, these products contained both P-1,4-glucan and P-1,3-glucan or only P-1,3-glucan in the presence of magnesium (Hayashi et al., 1987). The AN reagent has been used for isolation of cellulose (Updegraff, 1969), but unfortunately, P-1,4-glucan synthesized in vitro was found to be soluble in this reagent (Bacic and Delmer, 1981;Read and Delmer, 1991). Because of such reports in the literature and the difficulties in securing an ANIP, most investigators have not used the AN reagent in the assay for in vitro cellulose synthesis.In view of the lack of progress in accomplishing in vitro cellulose biosynthesis from higher plant extracts, we decided to use the valuable experience gained from the Acetobacter system and apply it to the cotton (Gossypium hirsutum) fiber. Based on numerous trials, we have found that about 4% of the total glucan isolated as the ANIP (Okuda et al., 1993) could be synthesized in vitro with an optimal combination of CB, cyclic nucleotide, magnesium, calcium, and digitonin. We also have found that the ANIP contains exclusively P-1,4-linked glucan, which has been identified as crystalline cellulose. In addition, approximately 25% of the total glucans is found as P-1,4-glucan and 69% as P-1,3-glucan in the EIP fraction synthesized under conditions favoring /3-1,4-glucan synthesis (Okuda et al., 1993). In addition, we have discovered that the EIP produced only with CB and calcium as effectors yielded essentially P-1,3-glucan (Okuda et al., 1993).The research described in this report provides new information for in vitro @-1,4-glucan synthesis from membrane fractions of cotton fibers, including data concerning putative activators, the combination of cofactors, and kinetic properties of P-1,3-and P-1,4-glucan synthases. From these results, we have gained new insight into the perennial and continuing problems underlying in vitro cellulose synthesis in higher plants.

Using differential product entrapment and photolabeling under specifying conditions, we identified a 37-kD polypeptide as the best candidate among the UDP-glucose-binding polypeptides for the catalytic subunit of cotton (Cossypium hirsutum) cellulose synthase. This polypeptide is enriched by entrapment under conditions favoring j3-1,4-glucan synthesis, and it is magnesium dependent and sensitive to unlabeled UDP-glucose. A 52-kD polypeptide was identified as the most likely candidate for the catalytic subunit of &1,3-glucan synthase because this polypeptide is the most abundant protein in the entrapment fraction obtained under conditions favoring &1,3-glucan synthesis, is coincident with B-1,3-glucan synthase activity, and is calcium dependent. The possible involvement of other polypeptides in the synthesis of &1,3-glucan is discussed.

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