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Cellulose as a biological sink of CO2
T. Inui, M. Anpo, K. Izui, S. Yanagida, T. Yamaguchi (Editors) Advances in Chemical Conversions for Mitigating Carbon Dioxide Studies in Surface Science and Catalysis, Vol. 114 9 1998 Elsevier Science B.V. All rights reserved.
243
Cellulose as a biological sink of C O 2 T. Hayashi, Y. Ihara, T. Nakai, T. Takeda, and R. Tominaga Wood Research Institute, Kyoto University, Gokasho, Uji, Kyoto 611, Japan
One strategy to enhance CO2 fixation is to increase the biological deposition of cellulose in woody plants, because cellulose which is the most abundant organic compound on the earth is made from CO2 through photosynthetic pathways in the walls of plant cells. Cellulose has a strong tendency to selfassociate into fibrils which are not easily hydrolyzed, either chemically or biologically, and accumulate in the walls. Certainly, cellulose is a good biological sink for CO2 on the earth, but the mechanism of cellulose biosynthesis is still unknown (the cellulose synthase activity in vitro in higher plants has not been completely identified or defined by anyone yet, and its gene is unknown [1]). In addition, the cellulose biosynthesis has not onlybeen identified and defined as chain polymerization but is also involved in a dynamism of cortical microtubule association during the developmental growth of woody plants. We report here our lab update on cellulose biosynthesis in higher plants to improve woody plants by genetic engineering through studies on the biosynthetic mechanism. 1. CELLWALL LOOSENING The plasma membranes of growing plant cells select to incorporate sugars, amino acids, ions and other low molecular weight compounds from the apoplastic space, and then, the cells have a certain level of osmotic pressure. The difference between their osmotic pressure and their wall pressure (=turgor pressure) is due to motive power (suction force) to suck water from the apoplastic space (Figure 1). The plant hormone auxin, which decreases the wall pressure in a growing plant cell, therefore induces cell elongation or expansion.
Wall pressure (Turgor pressure)
Osmotic pressure Suction force = OP--- WP Figure 1. The relationship between osmotic pressure (OP), wall pressure (WP), and suction force.
244 The p h e n o m e n o n is called cell wall loosening [2], in which the wall controls plant cell growth. The cell enlargement (elongation and/or expansion) substantially associates cellulose deposition during development. Xyloglucans probably contribute to the cross-linking of each cellulose microfibril network in the walls of growing plant cells [3]. The binding of adjacent microfibrils probably gives cell wall its rigidity. The cross-linking between perpendicular fibrils may function as a bracket, and that between parallel fibrils as a beam. The primary growth-promoting action of auxin is identified and defined by the cell wall modification inv olv ed in the xyloglucan solubilization in the walls of growing plant cells [3, 4], because the solubilization may cause the weakening of the wall which allows it to stretch, and subsequent turgor-driven wall expansion [5]. A u x i n - a n d / o r acid-induced growth has been proven to be accompanied by xyloglucan solubilization in the apoplastic space of Pisum sativ u m [6] and in poplar and soybean cells in suspension culture [7, 8], and with changes in xyloglucan molecular weights in higher plants [9]. Although there are several enzyme candidates responsible for xyloglucan turnover, i.e., xyloglucanase [10], xyloglucan endotransglycosylase (XET) [11, 12], expansin [13] and cellulase [14], the mechanism of xyloglucan turnover has not yet been clarified. The overexpression of the sense or antisense m R N A might help reveal the function of each enzyme in plant tissues. 2. DEPOSITION OF CELLULOSE
2.1.
Cellulose synthase Random sequencing of 1,000 clones from the cDNA library of the fiber cells revealed 750 clones of DNA sequences [15], which were computor-simulatively translated according to their nudeotide sequences and their potential 4,500 polypeptides in deduced amino acid sequences were subjected to a homology search with Acetobacter xylinum cellulose 4-~-glucosyltransferase [16]. The full length cDNAs of pcs A1 and pcs A2 have been obtained by using 5'-RACE m e t h o d [17] and sequenced. The cotton pcs A1 which appears to be a full length clone of 3,228 bp contains an open reading frame of 2,934 bp that encodes a polypeptide of 978 amino acids with a calculated molecular mass of about 110 kDa as cel A 1 shown by Pear et al. [18]. The cotton pcs A2 which appears to be a full length clone of 3,311 bp contains an open reading frame of 3,120 bp that encodes a polypeptide of 1,039 amino acids with a calculated molecular mass of about 125 kDa. Each deduced amino acid sequence contains one consensus sequence for UDP-glucose binding motif (Table 1). The cellulose 4-~-glucosyltransferase of Acetobacter x y l i n u m exhibits 42.8 % identity at the DNA level and 26.2 % identity at the whole deduced amino acid level to the pcs A2 polypeptide. The cotton cel A1 polypeptide exhibits 53.9 % identity at the DNA level and 68.7 % identity at the amino acid level to the pcs A2 polypeptide. The hydropathy profiles suggest at least two transmembrane helices, e.g., one is located in the N-terminal region and one is in C-terminal region. The central regions of the polypeptides are rather hydrophilic and are
245 probably catalytic sites in the cytoplasm. The hydrophilic regions have the conserved UDP-glucose binding motif which has been believed to bind to the substrate and to catalyze the transfer of glucose into pre-formed 1,4-~glucan. Table 1 Characterization of pcs A2 cDNA and its deduced amino acid sequence Length
Identity with bcs A (%)
UDP-glucose-
(bp)
Nucleotide Amino acid
binding motif
Gene
pcs A2 pcs A 1 bcs A
2.2.
Source
3,311 3,228 2,262
42.8 42.4 100
26.6 25.4 100
YPVEKVCCYVSDDG Cotton YPVDKVSCYISDDG Cotton WPPDKVNVYII.DDG Acetobacter
Formation of UDP-glucose Higher plants have two systems for the formation of UDP-glucose with UDP-glucose pyrophosphorylase (EC 2.7.7.9) and sucrose synthase (EC 2.4.1.13), although bacteria contain only one system (Figure 2). The sucrose synthase catalyzes the reaction: UDP-glucose + fructose = sucrose + UDP, a freely reversible reaction. The a m o u n t of the enzyme is much higher in nonphotosynthetic tissues, where sucrose is the source of carbon that is Figure 2. Formation of UDP-glucose translocated and cleaved by the in higher plants. enzyme to produce UDP-glucose for synthesis of cellulose as a major sink in plants. Therefore, the enzyme may function to produce UDP-glucose rather than to synthesize sucrose in plant tissues. UDP formed from UDP-glucose by 4-~-glucosyltransferase reactions can be recycled in a short time to produce UDPglucose by sucrose synthase. The production of UDP-glucose by the enzyme is a method of conserving energy ATP [18. 19], which only occurs in higher plants. In developing cotton fibers, the sucrose synthase, localized in arrays that parallel the helical pattern of cellulose deposition, may participate in the biosynthesis of cellulose [20]. The m u n g bean (Vigna radiata, Wilczek) sucrose synthase is a tetramer corn posed of identical subunits of 95 kDa, and its cDNA contains an open reading frame of 2,415 bp that encodes a polypeptide of 805 amino acids with a calculated molecular mass of 92,087 daltons. The recombinant sucrose synthase expressed
246 in Escherichia coli harboring an expression plasmid containing m u n g bean sucrose synthase cDNA conserves the activity of sucrose synthase [21]. 3.
ORIENTATION OF MICROFIBRILS BY CORTICAL MICROTUBULES
In higher plants, the microtubules have two functions, one is to determine the plane of cell division by the formation of the mitotic spindle, and the other is to orient the deposition of cellulose microfibrils by the assembly of microtubules in growing cells [22]. In fact, in growing plant cells, cellulose microfibrils are mostly transversely oriented against an elongating or expanding direction as a result of microtubule reorganization (Figure 3). However, this has been shown onlyby com paring the assembly of cortical microtubules with the orientation of microfibrils in the freeze fracture micrographs. To examine the interaction between cortical microtubules and microfibrils more directly, we prepared an isolated plasma membrane sheet Figure 3. The orientation of cellulose with cortical microtubules from microfibrils in elongating plant cells. tobbaco cells and demonstrated that ~glucan synthases penetrating through the membrane move in the fluid m e m b r a n e along cortical microtubules, forming microfibrils. In the presence of UDP-glucose, ~-glucan microfibrils were formed abundantly in the interface between the prepared membrane sheet and a polylysine-coated coverslip. The microfibrils appeared to be formed as short fibers at m a n y loci in the presence of taxol within a few minutes after the start of incubation, and longer fibers were formed after incubation for 30 min. The microfibrils formed during incubation were arranged closely in parallel to the microtubules. The rate of ~glucan elongation directly determined on the exoplasmic surface was 1.288 ~tm per min. W h e n the ordered structure of microtubules was disrupted by the treatment with propyzamide during the preparation of protoplasts, ~-glucans were deposited in masses on the prepared membrane sheet not in arrays. This suggests that the arrayed cortical microtubules are required for the formation of arranged microfibrils on the prepared membrane sheet.
247 4.
C A N WE IMPROVE TREES BY OVEREXPRESSION OF THE GENES?
One strategy to the enhance CO2 fixation in woody plants is to enhance the expression of genes required for cellulose deposition, which should enhance plant growth either by cell wall or just cellulose deposition. W e have already isolated the full length of cDNAs for three kinds of cellulases, XET and expansin as plant cell growth regulators, and for two kinds of cellulose synthases and sucrose synthase as a system of cellulose synthesis, as summarized in Table 2. If cellulose deposition is increased by the increased activity Of each enzyme in the transformants of Arabidopsis, the gene can be introduced to woody plants to determine the cellulose deposition. Each cDNA fragment was redoned into the binary plasmid pBE2113 containing a chimeric promoter E12~ [23]. Arabidopsis plants were transformed by the vacuum infiltration method [24]. Transform ant seeds were selected in the presence of 100 , g / m l kanamycin. Table 2 Potential cDNAs for enzymes responsible for cellulose deposition Molecular size
Length (bp)
Enzyme
Plant origin (kDa)
cDNA
ORF
Growth regulation Cellulase Cellulase Cellulase (EGL1) XET Expansin
55 56 54 34 28
1,580 1,550 1,473 1,341 779
1,482 1,518 1,458 879 774
Poplar Pea Pea Pea Pea
Cellulose synthesis Pcs I Pcs 2 Sucrose synthase
110 125 92
3,228 3,311 2,652
2,934 3,120 2,445
Cotton Cotton Mung bean
The carbohydrate analysis of the transformants would be expected to reveal the changes in xyloglucan and cellulose because carbohydrates are genetically regulated to form in the cell and to be secreted into the wall. In fact, there is evidence that novel cell walls are produced by hybridization of plants [25]. The polysaccharides may be changed not only in amount but also in molecular weight, and therefore, the cell walls of the transform ants will differ from those
248 of wild plants. Such modification of the cell wall is the kind of cell-wall engineering that should make useful contributions to plant biotechnology.
REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
D.P. Delmer and Y. Amor, Plant Cell, 7 (1995) 987. Y. Masuda, Bot. Mag. Tokyo Special Issue, 1 (1978) 103. T. Hayashi, Annu. Rev. Plant Physiol. Plant Mol. Biol., 40 (1989) 139. J.M. Labavitch, Ann. Rev. Plant Physiol., 32 (1981) 385. C.W. Lloyd (ed.), The Cytoskeletal Basis of Plant Growth and Form, Academic, London, 1991. M.E. Terry, R.L. Jones and B.A. Bonner, Plant Physiol., 68 (1981)531. T. Hayashi and T. Takeda, Biosci. Biotech. Biochem., 58 (1994) 1707. T. Hayashi, Y. Kato and K. Matsuda, Plant Cell Physiol., 21 (1980) 1405. E.P. Lorences and I. Zarra, J. Exp. Bot., 38 (1987) 960. T. Matsumoto, F. Sakai and T. Hayashi, Plant Physiol., 114 (1997) 661. S.C. Fry, R.C. Smith, K.F. Renwick, D.J. Martin, S.K. Hodge, K.J. Matthews, Biochem. J., 282 (1992) 821. K. Okazawa, Y. Sato, T. Nakagawa, K. Asada, I. Kato, E. Tomita and K. Nishitani, J. Biol. Chem., 268 (1993) 25364. D.J. Cosgrove, Plant Cell, 9 (1997) 1031. Y. Ohmiya, T. Takeda, S. Nakamura, F. Sakai and T. Hayashi, Plant Cell Physiol., 36 (1995) 607. N. Shiraishi (ed.), Kyoto Conference on Cellulose, The Cellulose Society of Japan, Kyoto, 1994. H.C. Wong, et al., Proc. Nail. Sci. USA, 87 (1990) 8130. M.A. Frohman, M.K. Dush and G.R. Martin, Proc. Natl. Sci. USA, 85 (1988) 8998. J.R. Pear, Y. Kawagoe, W.E. Schreckengost, D.P. Delmer and D.M. Stalker, Proc. Natl. Sci. USA, 93 (1996) 12637. P.S. Chourey and O.E. Nelson, Biochem. Gen., 14 (1976) 1041. Y. Amor, C.H. Haigler, S. Johnson, M. Wainscott and D.P. Delmer, Proc. Natl. Acad. Sci. USA, 92 (1995) 9353. T. Nakai, N. Tonouchi, T. Tsuchida, H. Mori, F. Sakai and T. Hayashi, Biosci. Biotech. Biochem., (1997)in press. R.J. Cyr, Annu. Rev. Cell Biol., 10 (1994) 153. I. Mitsuhara, et al., Plant Cell Physiol., 37 (1996) 49. I. Potrykus and G. Spangenberg (eds.), Gene Transfer to Plants, Springer, Berlin, 1995. C. Ohsumi and T. Hayashi, Biosci. Biotech. Biochem., 58 (1994) 959.
243
Cellulose as a biological sink of C O 2 T. Hayashi, Y. Ihara, T. Nakai, T. Takeda, and R. Tominaga Wood Research Institute, Kyoto University, Gokasho, Uji, Kyoto 611, Japan
One strategy to enhance CO2 fixation is to increase the biological deposition of cellulose in woody plants, because cellulose which is the most abundant organic compound on the earth is made from CO2 through photosynthetic pathways in the walls of plant cells. Cellulose has a strong tendency to selfassociate into fibrils which are not easily hydrolyzed, either chemically or biologically, and accumulate in the walls. Certainly, cellulose is a good biological sink for CO2 on the earth, but the mechanism of cellulose biosynthesis is still unknown (the cellulose synthase activity in vitro in higher plants has not been completely identified or defined by anyone yet, and its gene is unknown [1]). In addition, the cellulose biosynthesis has not onlybeen identified and defined as chain polymerization but is also involved in a dynamism of cortical microtubule association during the developmental growth of woody plants. We report here our lab update on cellulose biosynthesis in higher plants to improve woody plants by genetic engineering through studies on the biosynthetic mechanism. 1. CELLWALL LOOSENING The plasma membranes of growing plant cells select to incorporate sugars, amino acids, ions and other low molecular weight compounds from the apoplastic space, and then, the cells have a certain level of osmotic pressure. The difference between their osmotic pressure and their wall pressure (=turgor pressure) is due to motive power (suction force) to suck water from the apoplastic space (Figure 1). The plant hormone auxin, which decreases the wall pressure in a growing plant cell, therefore induces cell elongation or expansion.
Wall pressure (Turgor pressure)
Osmotic pressure Suction force = OP--- WP Figure 1. The relationship between osmotic pressure (OP), wall pressure (WP), and suction force.
244 The p h e n o m e n o n is called cell wall loosening [2], in which the wall controls plant cell growth. The cell enlargement (elongation and/or expansion) substantially associates cellulose deposition during development. Xyloglucans probably contribute to the cross-linking of each cellulose microfibril network in the walls of growing plant cells [3]. The binding of adjacent microfibrils probably gives cell wall its rigidity. The cross-linking between perpendicular fibrils may function as a bracket, and that between parallel fibrils as a beam. The primary growth-promoting action of auxin is identified and defined by the cell wall modification inv olv ed in the xyloglucan solubilization in the walls of growing plant cells [3, 4], because the solubilization may cause the weakening of the wall which allows it to stretch, and subsequent turgor-driven wall expansion [5]. A u x i n - a n d / o r acid-induced growth has been proven to be accompanied by xyloglucan solubilization in the apoplastic space of Pisum sativ u m [6] and in poplar and soybean cells in suspension culture [7, 8], and with changes in xyloglucan molecular weights in higher plants [9]. Although there are several enzyme candidates responsible for xyloglucan turnover, i.e., xyloglucanase [10], xyloglucan endotransglycosylase (XET) [11, 12], expansin [13] and cellulase [14], the mechanism of xyloglucan turnover has not yet been clarified. The overexpression of the sense or antisense m R N A might help reveal the function of each enzyme in plant tissues. 2. DEPOSITION OF CELLULOSE
2.1.
Cellulose synthase Random sequencing of 1,000 clones from the cDNA library of the fiber cells revealed 750 clones of DNA sequences [15], which were computor-simulatively translated according to their nudeotide sequences and their potential 4,500 polypeptides in deduced amino acid sequences were subjected to a homology search with Acetobacter xylinum cellulose 4-~-glucosyltransferase [16]. The full length cDNAs of pcs A1 and pcs A2 have been obtained by using 5'-RACE m e t h o d [17] and sequenced. The cotton pcs A1 which appears to be a full length clone of 3,228 bp contains an open reading frame of 2,934 bp that encodes a polypeptide of 978 amino acids with a calculated molecular mass of about 110 kDa as cel A 1 shown by Pear et al. [18]. The cotton pcs A2 which appears to be a full length clone of 3,311 bp contains an open reading frame of 3,120 bp that encodes a polypeptide of 1,039 amino acids with a calculated molecular mass of about 125 kDa. Each deduced amino acid sequence contains one consensus sequence for UDP-glucose binding motif (Table 1). The cellulose 4-~-glucosyltransferase of Acetobacter x y l i n u m exhibits 42.8 % identity at the DNA level and 26.2 % identity at the whole deduced amino acid level to the pcs A2 polypeptide. The cotton cel A1 polypeptide exhibits 53.9 % identity at the DNA level and 68.7 % identity at the amino acid level to the pcs A2 polypeptide. The hydropathy profiles suggest at least two transmembrane helices, e.g., one is located in the N-terminal region and one is in C-terminal region. The central regions of the polypeptides are rather hydrophilic and are
245 probably catalytic sites in the cytoplasm. The hydrophilic regions have the conserved UDP-glucose binding motif which has been believed to bind to the substrate and to catalyze the transfer of glucose into pre-formed 1,4-~glucan. Table 1 Characterization of pcs A2 cDNA and its deduced amino acid sequence Length
Identity with bcs A (%)
UDP-glucose-
(bp)
Nucleotide Amino acid
binding motif
Gene
pcs A2 pcs A 1 bcs A
2.2.
Source
3,311 3,228 2,262
42.8 42.4 100
26.6 25.4 100
YPVEKVCCYVSDDG Cotton YPVDKVSCYISDDG Cotton WPPDKVNVYII.DDG Acetobacter
Formation of UDP-glucose Higher plants have two systems for the formation of UDP-glucose with UDP-glucose pyrophosphorylase (EC 2.7.7.9) and sucrose synthase (EC 2.4.1.13), although bacteria contain only one system (Figure 2). The sucrose synthase catalyzes the reaction: UDP-glucose + fructose = sucrose + UDP, a freely reversible reaction. The a m o u n t of the enzyme is much higher in nonphotosynthetic tissues, where sucrose is the source of carbon that is Figure 2. Formation of UDP-glucose translocated and cleaved by the in higher plants. enzyme to produce UDP-glucose for synthesis of cellulose as a major sink in plants. Therefore, the enzyme may function to produce UDP-glucose rather than to synthesize sucrose in plant tissues. UDP formed from UDP-glucose by 4-~-glucosyltransferase reactions can be recycled in a short time to produce UDPglucose by sucrose synthase. The production of UDP-glucose by the enzyme is a method of conserving energy ATP [18. 19], which only occurs in higher plants. In developing cotton fibers, the sucrose synthase, localized in arrays that parallel the helical pattern of cellulose deposition, may participate in the biosynthesis of cellulose [20]. The m u n g bean (Vigna radiata, Wilczek) sucrose synthase is a tetramer corn posed of identical subunits of 95 kDa, and its cDNA contains an open reading frame of 2,415 bp that encodes a polypeptide of 805 amino acids with a calculated molecular mass of 92,087 daltons. The recombinant sucrose synthase expressed
246 in Escherichia coli harboring an expression plasmid containing m u n g bean sucrose synthase cDNA conserves the activity of sucrose synthase [21]. 3.
ORIENTATION OF MICROFIBRILS BY CORTICAL MICROTUBULES
In higher plants, the microtubules have two functions, one is to determine the plane of cell division by the formation of the mitotic spindle, and the other is to orient the deposition of cellulose microfibrils by the assembly of microtubules in growing cells [22]. In fact, in growing plant cells, cellulose microfibrils are mostly transversely oriented against an elongating or expanding direction as a result of microtubule reorganization (Figure 3). However, this has been shown onlyby com paring the assembly of cortical microtubules with the orientation of microfibrils in the freeze fracture micrographs. To examine the interaction between cortical microtubules and microfibrils more directly, we prepared an isolated plasma membrane sheet Figure 3. The orientation of cellulose with cortical microtubules from microfibrils in elongating plant cells. tobbaco cells and demonstrated that ~glucan synthases penetrating through the membrane move in the fluid m e m b r a n e along cortical microtubules, forming microfibrils. In the presence of UDP-glucose, ~-glucan microfibrils were formed abundantly in the interface between the prepared membrane sheet and a polylysine-coated coverslip. The microfibrils appeared to be formed as short fibers at m a n y loci in the presence of taxol within a few minutes after the start of incubation, and longer fibers were formed after incubation for 30 min. The microfibrils formed during incubation were arranged closely in parallel to the microtubules. The rate of ~glucan elongation directly determined on the exoplasmic surface was 1.288 ~tm per min. W h e n the ordered structure of microtubules was disrupted by the treatment with propyzamide during the preparation of protoplasts, ~-glucans were deposited in masses on the prepared membrane sheet not in arrays. This suggests that the arrayed cortical microtubules are required for the formation of arranged microfibrils on the prepared membrane sheet.
247 4.
C A N WE IMPROVE TREES BY OVEREXPRESSION OF THE GENES?
One strategy to the enhance CO2 fixation in woody plants is to enhance the expression of genes required for cellulose deposition, which should enhance plant growth either by cell wall or just cellulose deposition. W e have already isolated the full length of cDNAs for three kinds of cellulases, XET and expansin as plant cell growth regulators, and for two kinds of cellulose synthases and sucrose synthase as a system of cellulose synthesis, as summarized in Table 2. If cellulose deposition is increased by the increased activity Of each enzyme in the transformants of Arabidopsis, the gene can be introduced to woody plants to determine the cellulose deposition. Each cDNA fragment was redoned into the binary plasmid pBE2113 containing a chimeric promoter E12~ [23]. Arabidopsis plants were transformed by the vacuum infiltration method [24]. Transform ant seeds were selected in the presence of 100 , g / m l kanamycin. Table 2 Potential cDNAs for enzymes responsible for cellulose deposition Molecular size
Length (bp)
Enzyme
Plant origin (kDa)
cDNA
ORF
Growth regulation Cellulase Cellulase Cellulase (EGL1) XET Expansin
55 56 54 34 28
1,580 1,550 1,473 1,341 779
1,482 1,518 1,458 879 774
Poplar Pea Pea Pea Pea
Cellulose synthesis Pcs I Pcs 2 Sucrose synthase
110 125 92
3,228 3,311 2,652
2,934 3,120 2,445
Cotton Cotton Mung bean
The carbohydrate analysis of the transformants would be expected to reveal the changes in xyloglucan and cellulose because carbohydrates are genetically regulated to form in the cell and to be secreted into the wall. In fact, there is evidence that novel cell walls are produced by hybridization of plants [25]. The polysaccharides may be changed not only in amount but also in molecular weight, and therefore, the cell walls of the transform ants will differ from those
248 of wild plants. Such modification of the cell wall is the kind of cell-wall engineering that should make useful contributions to plant biotechnology.
REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
D.P. Delmer and Y. Amor, Plant Cell, 7 (1995) 987. Y. Masuda, Bot. Mag. Tokyo Special Issue, 1 (1978) 103. T. Hayashi, Annu. Rev. Plant Physiol. Plant Mol. Biol., 40 (1989) 139. J.M. Labavitch, Ann. Rev. Plant Physiol., 32 (1981) 385. C.W. Lloyd (ed.), The Cytoskeletal Basis of Plant Growth and Form, Academic, London, 1991. M.E. Terry, R.L. Jones and B.A. Bonner, Plant Physiol., 68 (1981)531. T. Hayashi and T. Takeda, Biosci. Biotech. Biochem., 58 (1994) 1707. T. Hayashi, Y. Kato and K. Matsuda, Plant Cell Physiol., 21 (1980) 1405. E.P. Lorences and I. Zarra, J. Exp. Bot., 38 (1987) 960. T. Matsumoto, F. Sakai and T. Hayashi, Plant Physiol., 114 (1997) 661. S.C. Fry, R.C. Smith, K.F. Renwick, D.J. Martin, S.K. Hodge, K.J. Matthews, Biochem. J., 282 (1992) 821. K. Okazawa, Y. Sato, T. Nakagawa, K. Asada, I. Kato, E. Tomita and K. Nishitani, J. Biol. Chem., 268 (1993) 25364. D.J. Cosgrove, Plant Cell, 9 (1997) 1031. Y. Ohmiya, T. Takeda, S. Nakamura, F. Sakai and T. Hayashi, Plant Cell Physiol., 36 (1995) 607. N. Shiraishi (ed.), Kyoto Conference on Cellulose, The Cellulose Society of Japan, Kyoto, 1994. H.C. Wong, et al., Proc. Nail. Sci. USA, 87 (1990) 8130. M.A. Frohman, M.K. Dush and G.R. Martin, Proc. Natl. Sci. USA, 85 (1988) 8998. J.R. Pear, Y. Kawagoe, W.E. Schreckengost, D.P. Delmer and D.M. Stalker, Proc. Natl. Sci. USA, 93 (1996) 12637. P.S. Chourey and O.E. Nelson, Biochem. Gen., 14 (1976) 1041. Y. Amor, C.H. Haigler, S. Johnson, M. Wainscott and D.P. Delmer, Proc. Natl. Acad. Sci. USA, 92 (1995) 9353. T. Nakai, N. Tonouchi, T. Tsuchida, H. Mori, F. Sakai and T. Hayashi, Biosci. Biotech. Biochem., (1997)in press. R.J. Cyr, Annu. Rev. Cell Biol., 10 (1994) 153. I. Mitsuhara, et al., Plant Cell Physiol., 37 (1996) 49. I. Potrykus and G. Spangenberg (eds.), Gene Transfer to Plants, Springer, Berlin, 1995. C. Ohsumi and T. Hayashi, Biosci. Biotech. Biochem., 58 (1994) 959.