Cellulose biosynthesis in seed plants involves rosette structures containing cellulose synthases located in the plasma membrane. Cellulose microfibrils are synthesized from the donor molecule UDP-glucose and deposited following the direction of underlying microtubules. While isolated plant membranes can synthesize callose when incubated with UDP-glucose, providing sucrose to intact cotton fibers results in the majority of the product being cellulose, suggesting sucrose synthase forms a complex channeling carbon from sucrose to UDP-glucose for cellulose synthesis. Bacteria like Gluconacetobacter xylinus are also able to synthesize cellulose extracellularly and have provided insights into the cellulose synthesis process.

1. Cellulose biosynthesis in seed plants

2. Essay title Please write this exact title on the front page of the essay: Critically discuss what is known about the biosynthesis of cellulose in seed plants.

3. Reviews (do NOT use these for seminars) Guerriero, G., Fugelstad, J. and Bulone, V. (2010) What do we really know about cellulose biosynthesis in higher plants? Journal of Integrative Plant Biology 52, 161-175. Bessueille, L. and V. Bulone. 2008. A survey of cellulose biosynthesis in higher plants. Plant Biotechnology. 25:315-322. Endler, A. and S. Persson. 2011. Cellulose synthases and synthesis in Arabidopsis . Molecular Plant. 4:199–211. Harris, D. and DeBolt, S. (2010) Synthesis, regulation and utilization of lignocellulosic biomass. Plant Biotechnology Journal 8 , 244-262. Wightman, R. and Turner, S. (2010) Trafficking of the plant cellulose synthase complex. Plant Physiology 153 , 427-432. Taylor, N.G. (2008) Cellulose biosynthesis and deposition in higher plants. New Phytologist 178 , 239-252. References on Cellulose Biosynthesis

4. Original papers Atanassov, I. I., Pittman, J. K., & Turner, S. R. (2009). Elucidating the mechanisms of assembly and subunit interaction of the cellulose synthase complex of Arabidopsis secondary cell walls. Journal of Biological Chememistry, 284 , 3833-3841. Barratt, D.H.P., Derbyshire, P., Findlay, K., Pike, M., Wellner, N., Lunn, J., Feil, R., Simpson, C., Maule, A.J. and Smith, A.M. (2009) Normal growth of Arabidopsis requires cytosolic invertase but not sucrose synthase. Proceedings of the National Academy of Science USA 106 13124-13129. Bessueille, L., Sindt, N., Guichardant, M., Djerbi, S., Teeri, T.T. and Bulone, V. (2009) Plasma membrane microdomains from hybrid aspen cells are involved in cell wall polysaccharide biosynthesis. Biochemical Journal 420 , 93-103. Bischoff, V., Nita, S., Neumetzler, L., Schindelasch, D., Urbain, A., Eshed, R., Persson, S., Delmer, D. and Scheible, W.-R. (2010) TRICHOME BIREFRINGENCE and its homolog AT5G01360 encode plant-specific DUF231 proteins required for cellulose biosynthesis in Arabidopsis. Plant Physiology 153 , 590-602. Bowling, A. J., & Brown Jr, R. M. (2008). The cytoplasmic domain of the cellulose-synthesizing complex in vascular plants. Protoplasma, 233 , 115-127. Cifuentes, C., Bulone, V. and Emons, A.M.C. (2010) Biosynthesis of callose and cellulose by detergent extracts of tobacco cell membranes and quantification of the polymers synthesized in vitro . Journal of Integrative Plant Biology 52 , 221-233. Coleman, H.D., Yan, J. and Mansfield, S.D. (2009) Sucrose synthase affects carbon partitioning to increase cellulose production and altered cell wall ultrastructure. Proceedings of the National Academy of Science USA 106 , 13118-13123. Crowell, E. F., Bischoff, V., Desprez, T., Rolland, A., Stierhof, Y.-D., Schumacher, K., Gonneau, M., Höfte, H., & Samantha Vernhettesa. (2009). Pausing of Golgi bodies on microtubules regulates secretion of cellulose synthase complexes in Arabidopsis . Plant Cell 21 , 1141-1154. Daras, G., Rigas, S., Penning, B., Milioni, D., McCann, M.C., Carpita, N.C., Fasseas, C. and Hatzopoulos, P. (2009) The thanatos mutation in Arabidopsis thaliana cellulose synthase 3 ( AtCesA3 ) has a dominant-negative effect on cellulose synthesis and plant growth. New Phytologist 184 , 114-126. Desprez, T., Juraniec, M., Crowell, E.F., Jouy, H., Pochylova, Z., Parcy, F., Hofte, H., Gonneau, M. and Vernhettes, S. (2007) Organization of cellulose synthase complexes involved in primary cell wall synthesis in Arabidopsis thaliana Proceedings of the National Academy of Sciences, USA 104 , 15572–15577. Fujii, S., Hayashi, T. and Mizuno, K. (2010) Sucrose synthase is an integral component of the cellulose synthesis machinery. Plant Cell Physiology 51 , 294-301. Desprez T, Juraniec M, Crowell EF, Jouy H, Pochylova Z, Parcy F, Hofte H, Gonneau M, Vernhettes S (2007) Organization of cellulose synthase complexes involved in primary cell wall synthesis in Arabidopsis thaliana. Proceedings of the National Academy of Sciences, USA 104: 15572–15577.

5. Gutierrez, R., Lindeboom, J. J., Paredez, A. R., Emons, A. M. C., & Ehrhardt, D. W. (2009). Arabidopsis cortical microtubules position cellulose synthase delivery to the plasma membrane and interact with cellulose synthase trafficking compartments. Nature Cell Biology 11 , 797-808. Hofmannová, J., Schwarzerová, K., Havelková, L., Boříková, P., Petrášek, J., & Opatrny, Z. (2008). A novel, cellulose synthesis inhibitory action of ancymidol impairs plant cell expansion. Journal of Experimental Botany, 59 , 3963-3974. Li, M., Xiong, G., Li, R., Cui, J., Tang, D., Zhang, Z., Pauly, M., Cheng, Z. and Zhou, Y. (2009) Rice cellulose synthase-like D4 is essential for normal cell-wall biosynthesis and plant growth. The Plant Journal 60 , 1055-1069. Maloney, V.J. and Mansfield, S.D. (2010) Characterization and varied expression of a membrane-bound endo-β-1,4-glucanase in hybrid poplar. Plant Biotechnology Journal 8 , 294-307 Paredez, A. R., Somerville, C. R., & Ehrhardt, D. W. (2006). Visualization of cellulose synthase demonstrates functional association with microtubules. Science, 312 , 1491-1495. Paredez, A. R., Persson, S., Ehrhardt, D. W., & Somerville, C. R. (2008). Genetic evidence that cellulose synthase activity influences microtubule cortical array organization. Plant Physiology, 147 , 1723-1734. Rajangam, A. S., Kumar, M., Aspeborg, H., Guerriero, G., Arvestad, Pansri, P., Brown, C. J.-L., Hober, S., Blomqvist, K., Divne, C., Ezcurra, I., Mellerowicz, E., Sundberg, B., Bulone, V., & Teeri, T. T. (2008). MAP20, a microtubule-associated protein in the secondary cell walls of hybrid aspen, is a target of the cellulose synthesis inhibitor 2,6-dichlorobenzonitrile. Plant Physiology, 148 , 1283-1294. Salnikov, V.V., Grimson, M.J., Delmer, D.P. and Haigler, C.H. (2001) Sucrose synthase localizes to cellulose synthesis sites in tracheary elements. Phytochemistry 57 , 823-833. Stork, J., Harris, D., Griffiths, J., Williams, B., Beisson, F., Li-Beisson, Y., Mendu, V., Haughn, G. and DeBolt, S. (2010) CELLULOSE SYNTHASE9 serves a nonredundant role in secondary cell wall synthesis in Arabidopsis epidermal testa cells. Plant Physiology 153 , 580-589. Takahashi, J., Rudsander, U.J., Hedenström, M., Banasiak, A., Harholt, J., Amelot, N., Immerzeel, P., Ryden, P., Endo, S., Ibatullin, F.M., Brumer, H., del Campillo, E., Master, E.R., Scheller, H.V., Sundberg, B., Teeri, T.T. and Mellerowicz, E.J. (2009) KORRIGAN1 and its aspen homolog PttCel9A1 decrease cellulose crystallinity in Arabidopsis stems Plant and Cell Physiology 50 , 1099-1115. Timmers, J., Samantha Vernhettes, S., Desprez, T., Vincken, J.-P., Visser, R. G. F., & Trindade, L. M. (2009). Interactions between membrane-bound cellulose synthases involved in the synthesis of the secondary cell wall. FEBS Letters, 583 , 978-982. Wightman, R., Marshall, R., & Turner, S. R. (2009). A cellulose synthase-containing compartment moves rapidly beneath sites of secondary wall synthesis. Plant Cell Physiology, 50 , 584-594. Wightman, R., & Turner, S. R. (2008). The roles of the cytoskeleton during cellulose deposition at the secondary cell wall. Plant Journal, 54 , 794-805.

6. Cellulose is a (1  4)-  - D -glucan

7. Occurs in the form of microfibrils In primary walls dimensions 3-3.5 nm wide crystallites (~ 25 cellulose molecules 5 x 5, rather than 36)

8. From: Brett & Waldron (1996) Physiol. & biochemistry of plant cell walls, p 78 Synthesized from an activated donor: UDP-glucose

9. Location of synthesis – plasma membrane Studies with the transmission electron microscope (TEM) of the plasma membranes of all land plants after preparation using freeze fracturing showed rosettes of 6 particles From: Delmer, D. P. (1999). Annual Review Plant Physiology Plant Molecular Biology, 50, 245-276.

10. Rosettes are often located at the ends of cellulose microfibrils Particles of rosette contain cellulose synthases (immunogold labelling) From: Taiz and Zeiger (2006) Plant Physiology 4 th edition. Sinauer, Sunderland (USA).

11. Direction of cellulose microfibrils follows that of underlying microtubules From: Lloyd, C. (2006). Science, 312, 1482-1483. Heath, I. B. (1974). A unified hypothesis for the role of membrane bound enzyme complexes and microtubules in plant cell wall synthesis Journal of Theoretical Biology 48, 445-449. Paredez et al. (2006). Science, 312 , 1491-1495.

12. But when plasma membranes or mixed membranes are isolated from developing cotton fibres and incubated with UDP-glucose, labelled ( 14 C or 3 H), label is mostly incorporated in another polysaccharide: callose [(1  3)-  -glucan]. [Biochemical approach]

13. Delmer (1995) showed that if feed 14 C-sucrose to developing cotton fibres 'permeabilized' with digitonin (a detergent): - Up to 60% of product cellulose - Rates of synthesis much higher than usual, approach in vivo rates. (Amor et al. 1995 PNAS USA 92, 9353-9357) At least half of sucrose synthase (SuSy) in cotton fibres tightly associated with plasma membrane. Catalyses: Sucrose + UDP  UDP-Glc + Fructose Suggested SuSy forms a complex with cellulose synthase and channels C from sucrose to UDP-Glc to cellulose.

15. Cellulose synthesis in bacteria Several genera of bacteria synthesize cellulose Gluconacetobacter xylinus (formerly Acetobacter xylinus) synthesizes massive amounts of extracellular cellulose as a pellicle. Used as a model organism. Unlike cellulose synthases of seed plants, can solubilize cellulose synthases with digitonin and obtain high rates of in vitro cellulose synthesis.

16. If solublized enzyme is incubated in vitro with UDP-Glc, cellulose fibrils visible in TEM form within 20-30 min. Operon of 4 genes involved in cellulose synthesis in A. xylinum Genes have been cloned: first (A) encodes catalytic subunit; second (B) encodes a regulatory subunit; third (C) function not clear could encode a pore-forming protein; and fourth (D) encodes a protein that may control crystallization of the microfibrils.

17. Do similar genes occur in angiosperms? Attempts to identify these using antibodies and nucleic acid hybridization probes failed Pear et al. (1996) PNAS USA 93, 12637-12642 Used a bioinformatic approach. Identified 2 cDNAs from a cotton ( Gossypium hirsutum ) fibre cDNA library 30% sequence similarity to G. xylinus gene A. Expressed abundantly at onset of secondary wall formation: GhCESA1 and GhCESA2 Definitive genetic evidence for functionality demonstrated using an Arabidopsis thaliana r adial sw elling mutant ( rsw mutants) (Arioli et al. 1998 Science, 279, 717-720). Temperature sensitive mutants: root-tip swelling at 31  , but not at 18  C

18. Wild type rsw1 From: Baskin et al. (1992) Australian Journal of Plant Physiology, 19, 427-437 .

19. rsw1 mutant reduced amount of cellulose synthesized & reduced cellulose crystallinity RSW1 cloned & found to complement rsw1 mutant Restored the wildtype – functionally a cellulose synthase ( AtCESA1 ) In rsw1 at 31°C, single particles rather than rosettes In mutant, alanine substitutes for valine at position 549.

20. Cellulose synthase superfamily 10 Cellulose synthase genes in A. thaliana ( AtCESA1-10 ), plus 30 cellulose synthase-like ( CSL ) genes (cellulose synthase superfamily) Fincher, G. B. (2009). Plant Physiology, 149 , 27-37.

21. Structures of predicted CESA proteins Vertical bars: predicted transmembrane domains (2 near N-terminal region and 6 near the C-terminus) CESAs from bacteria and angiosperms characterized by 4 conserved domains (U1-U4) U1-U3 each have a conserved aspartic acid residue (D) U4 contains a conserved QXXRW motif (Q glutamine; R arginine; W tryptophan) The D,D,D,QXXRW motifs are characteristic of family 2 β -glycosyltransferases (Carbohydrate active enzymes server at: http://afmb.cnrs-mrs.fr/CAZY/ ) In CESAs they occur in the cytoplasmic domain and bind UDP-glucose

22. From: Delmer, D. P. (1999). Annual Review Plant Physiology Plant Molecular Biology, 50, 245-276.

23. Guerriero, G., Fugelstad, J. and Bulone, V. (2010) Journal of Integrative Plant Biology 52, 161-175 .

24. Structures of predicted CESA proteins Compared with CESAs from bacteria, those from angiosperms have: Zinc finger domain (Zn) Hypervariable region (HVR1) Plant-conserved region (CR-P) Class-specific region (CSR) also known as hypervariable region 2 Callose synthase is encoded by glucan synthase-like ( GSL ) genes

25. Numerous other cellulose synthase mutants in A. thaliana e.g. ir regular x ylem mutants ( irx ) with collapsed xylem cells e.g. isoxaben (a herbicide) resistant ( ixr ) Mutant, antisense and expression analyses of the 10 AtCESA genes suggests that at least 3 encode proteins that synthesize cellulose in cells laying down primary walls: AtCESA1 , 3 , & one of the AtCESA6 -related CESA s (2, 5, 6 or 9). And at least 3 encode proteins that synthesize cellulose in cells laying down secondary walls: AtCESA4 , 7 & 8

26. From: Somerville (2006). Annual Review of Cell and Developmental Biology, 22, 53-78 .

27. Seminars Only 10 minutes, including questions Select an original, experimental paper (NOT a review) from the list We will try to do all the seminars in one session (we can continue Session 1 in Mac 2 (11-12 noon) Use PowerPoint and load file on to the hard drive at the beginning of the session Focus on the Results section. The Introduction should be no longer than one slide