Vitro Cellulose Synthesis

Attempts to synthesize cellulose in vitro by using detergent solubilized enzymes frequently lead to the accumulation of callose, a linear (1 3)- 3-D-glucan (Delmer 1987 Okuda et al. 1993). Even in the few cases where successful in vitro cellulose synthesis has been demonstrated, i.e., in blackberry (Lai Kee Him et al. 2002), cotton and mung bean (Kudlicka et al. 1995 Kudlicka et al. 1996 Kudlicka and 

Professor Vincent Bulone and Dr. Johan Edqvist are acknowledged for valuable input and critical reading of the manuscript. This work was supported by the European Commission grant QLK5-CT2001-00443. 

Allona I., Quinn M., Shoop E., Swope K., St Cyr S., Carlis J., RiedI J., Retzel E., Campbell M.M., Sederoff R., and Whetten R.W. 1998. Analysis of xylem formation in pine by cDNA sequencing. 

Andersson-Gunneras S., Mellerowicz E.J., Love J., Segerman B., Ohmiya Y, Coutinho P.M., Nilsson R, Henrissat B., Moritz T., and Sundberg B. 2006. Biosynthesis of cellulose-enriched tension wood in Populus global analysis of transcripts and metabolites identifles biochemical and developmental regulators in secondary wall biosynthesis. Plant J 45 144-165. 

Amino S., Takeuchi Y, and Komamine A. 1985. Changes in the enzyme activities involved in formation and interconversion of UDP-sugars during cell cycle in a synchronous culture of Catharanthus roseus. Physiol Plant 64 111-117. 

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Kudlicka K., Brown, Jr. R.M. Li L., Lee J.H., Shin H., and Kuga S. 1995. P-Glucan synthesis in the cotton fiber. IV. In vitro assembly of the cellulose I allomorph. Plant Physiol 107 111-123. Kudlicka K., Lee J.H., and Brown, Jr. R.M., 1996. A comparative analysis of in vitro cellulose synthesis from cell-free extracts of mung bean (Vigna radiata, Fabaceae) and cotton (Gossypium hirsutum, Malvaceae). Am J Bot 83 274-284. 

ICudlicka K., Lee J.H., and Brown, Jr. R.M., 1996. A comparative analysis of in vitro cellulose synthesis from cell-free extracts of mung bean (Vigna radlata, Fabaceae) and cotton Gossypium hirsutum, Malvaceae). Am J Bot 83 274-284. 

For the cellulose synthesis, guanosine diphosphate probably occurs in place of UDP. Adenosine triphosphate or Cori ester is also used for in vitro... 

Jasmonic acid-induced tuberization appears to be due to its being capable of inducing expansion of potato cells rather than the division of cells in vitro. The expansion-inducing activity seems to be specific to jasmonic acid and related compounds since none of the other plant hormones tested have appreciable activity [57]. Further studies [58] showed that jasmonic acid can increase the levels of sucrose and cell wall polysaccharides in potato cells in vitro. The cellulose synthesis inhibitor 2,6-dichlorobenzonitrile inhibited the jasmonic-acid-induced expansion of potato cells. These results support the finding that cell wall polysaccharide and the accumulation of sucrose are involved in jasmonic-acid-induced expansion of cells. Disorganizers of microtubules such as colchicine and isopropyl N-phenyl-carbamate and inhibitors of microfilaments, such as cytochalasin, strongly inhibited the jasmonic-acid-induced expansion of potato cells, suggesting the involvement of a cytoskeletal structure in the expansion of cells by this compound. 

Aloni Y, Delmer D.P., and Benziman M. 1982. Achievement of high rates of in vitro synthesis of 1, 4-beta-D-glucan activation by cooperative interaction of the Acetobacter xylinum enzyme system with GTP, polyethylene glycol, and a protein factor. Proc Natl Acad Sci USA 79(21) 6448-6452. Amikam D. and Benziman M. 1989. Cyclic diguanylic acid and cellulose synthesis in Agrobacterium tumefaciens. J Bacterid 171(12) 6649-6655. 

TCs (Delmer 1999 Roberts et al. 2002), these domains are obvious candidates for playing a role in particle association in rosettes. This hypothesis is supported by the results of in vitro assays that directly implicate the zinc-binding domain in rosette assembly (Kurek et al. 2002). Terminal complex dissociation in response to cellulose synthesis inhibitors (Mizuta and Brown, Jr. 1992 Peng et al. 2001 Kiedaisch et al. 2003) suggest that the particles that compose linear and rosette TCs are held together in different ways. One of these inhibitors (AE FI 50944) is effective in organisms with rosettes, but not linear TCs (Kiedaisch et al. 2003), whereas another (dichlorobenzonitrile) disrupts the linear TCs of Vaucheria hamata (Mizuta and Brown, Jr. 1992). 

Kirretic irrformatiorr orr cellulose synthesis is scarce in the literature. The in vitro studies reported are limited almost exclusively to experimental systems, such as bacteria and elongating cotton and flax fibers (Alorri et al. 1982 Delmer et al. 1993 Li et al. 1993). Li et al. (1993), analyzing cellulose synthase activity in cotton fibers, reported the and for UDP-glucose to be respectively 0.4 mM and 2.8 rrmol min mg protein. If cellulose synthase exhibits similar properties in vivo then it must operate under substrate-saturated conditions since the concentration of UDP-glucose in the cytosol is 13 mM (Dancer et al. 1990 Krause and Stitt 1992 Winter et al. 1993,1994). 

True estimates of for cellulose synthase, however, are still problematic. Delmer (1993, personal communications), for example, has noted that the rates for cellulose synthesis in vitro reported by Li et al. (1993) were no more than 5% of those occurring in vivo during primary wall synthesis. 

Altogether, these data indicate that the conditions for in vitro synthesis of cellulose must be optimized for each plant species and that there is no general recipe that can be applied regardless of the source of enzyme and conditions of extraction from the plasma membrane. So far, the only factor that is common to the protocols that have led to the highest in vitro synthesis of cellulose is the use of Mops buffer. In particular, in studies on the blackberry (Lai Kee Him et al. 2002), cotton fiber (Kudlicka et al. 1996 Peng et al. 2002) and mung bean enzymes (Kudlicka et al. 1996), as well as in our recent work on hybrid aspen (Colombani et al. 2004), Mops has been described as the buffer of choice over the previously used Tris to improve the yields of in vitro cellulose. The conditions that have led to successful in vitro syntheses of cellulose are summarized in Table 8-1. 

Pillonel C. and Meier H. 1985. Influence of external factors on callose and cellulose synthesis during incubation in vitro of intact cotton fibers with [ " CJsucrose. Planta 165 76-84. 

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