, Volume 18, Issue 2, pp 371–384 | Cite as

Isolation and characterization of the cellulose synthase genes PpCesA6 and PpCesA7 in Physcomitrella patens

  • Hua Zhang Wise
  • Inder M. Saxena
  • R. Malcolm BrownJr.Email author


Analysis of cellulose biosynthesis using molecular approaches has been successful in identifying genes in many cellulose-producing organisms, yet the mechanism of cellulose biosynthesis still remains to be understood. We are interested in developing the moss Physcomitrella patens as a useful system for the study of cellulose biosynthesis. This moss affords a number of advantages including a haploid dominated gametophyte and a very high efficiency of homologous recombination in its nuclear DNA for constructing gene knockouts. In addition, P. patens has only a primary cell wall unlike Arabidopsis thaliana, which has both a primary and a secondary cell wall. We identified two full-length cellulose synthase (CesA) genes of P. patens, PpCesA6 and PpCesA7 from an EST database and have analyzed the genomic sequences. PpCesA6 and PpCesA7 show high similarity to each other, both at the cDNA and genomic DNA levels. Single and double knockouts of PpCesA6 and PpCesA7 were generated and screened for phenotypic changes. While the PpCesA6 and PpCesA7 single knockouts did not show any obvious phenotypic differences from the wild-type, the double knockout had significantly reduced stem length. These results suggest that PpCesA6 and PpCesA7 probably have a very similar role in cellulose biosynthesis and their functions may be redundant. Additionally, their roles may overlap with the other P. patens CesAs as observed for CesAs involved in primary cell wall biosynthesis in A. thaliana.


Cellulose synthase CesA Cellulose biosynthesis Homologous recombination Physcomitrella patens 



We would like to thank Dr. Ralph Quatrano at the University of Washington for kindly providing us P. patens protonemata and RIKEN BioResource Center, Japan for providing us with the EST clones. We would also like to thank Dr. Rumiko Kofuji for giving us permission to use the pGFPmutnptII vector, Dr. Tomaki Nishiyama for giving us permission to use the pTN3 vector, Dr. Yuji Hiwatashi for giving us permission to use the p35S-Zeo vector and RIKEN BioResource Center, Japan for providing us with these vectors.


  1. Appenzeller L, Doblin M, Barreiro R, Wang HY, Niu XM, Kollipara K, Carrigan L, Tomes D, Chapman M, Dhugga KS (2004) Cellulose synthesis in maize: isolation and expression analysis of the cellulose synthase (CesA) gene family. Cellulose 11:287–299CrossRefGoogle Scholar
  2. Arioli T, Peng L, Betzner AS, Burn J, Wittke W, Herth W, Camilleri C, Hofte H, Plazinski J, Birch R, Cork A, Glover J, Redmond J, Williamson RE (1998) Molecular analysis of cellulose biosynthesis in Arabidopsis. Science 279:717–720CrossRefGoogle Scholar
  3. Ashton NW, Champagne CEM, Weiler T, Verkoczy LK (2000) The bryophyte Physcomitrella patens replicates extrachromosomal transgenic elements. New Phytol 146:391–402CrossRefGoogle Scholar
  4. Blanton RL, Fuller D, Iranfar N, Grimson MJ, Loomis WF (2000) The cellulose synthase gene of Dictyostelium. Proc Natl Acad Sci USA 97:391–2396CrossRefGoogle Scholar
  5. Brown RM Jr, Montezinos D (1976) Cellulose microfibrils: visualization of biosynthetic and orienting complexes in association with the plasma membrane. Proc Natl Acad Sci USA 73:143–147CrossRefGoogle Scholar
  6. Brown RM Jr (1990) Algae as tools in studying the biosynthesis of cellulose: nature’s most abundant macromolecule. In: Wiessner W, Robinson DG, Starr RC (eds) Experimental phycology: cell walls and surfaces, reproduction, photosynthesis. Spring-Verlag, New York, pp 19–39Google Scholar
  7. Brown RM Jr, Willison JHM, Richardson CL (1976) Cellulose biosynthesis in Acetobater xylium: visualization of the site of synthesis and direct measurement of the in vivo process. Proc Natl Acad Sci USA 73:4565–4569CrossRefGoogle Scholar
  8. Carpita NC, Gibeaut DM (1993) Structural models of the primary cell walls in flowering plants: consistency of molecular structure with the physical properties of the walls during growth. Plant J 3:1–30CrossRefGoogle Scholar
  9. Chiu W, Niwa Y, Zeng W, Hirano T, Kobayashi H, Sheen J (1996) Engineered GFP as a vital reporter in plants. Curr Biol 6:325–330CrossRefGoogle Scholar
  10. Cove DJ, Knight CD, Lamparter T (1997) Mosses as model systems. Trends Plant Sci 2:99–105CrossRefGoogle Scholar
  11. Desprez T, Vernhettes S, Fagard M, Refregier G, Desnos T, Aletti E, Pelletier N, Py S, Hofte H (2002) Resistance against herbicide isoxaben and cellulose deficiency caused by distinct mutations in same cellulose synthase isoform CESA6. Plant Physiol 128:482–490CrossRefGoogle Scholar
  12. 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. Proc Natl Acad Sci USA 104:15572–15577CrossRefGoogle Scholar
  13. Djerbi S, Lindskog M, Arvestad L, Sterky F, Teeri TT (2005) The genome sequence of black cottonwood (Populus trichocarpa) reveals 18 conserved cellulose synthase (CesA) genes. Planta 221:739–746CrossRefGoogle Scholar
  14. Franz G, Blaschek W (1990) Cellulose. In: Dey PM, Harborne JB (eds) Methods in plants biochemistry, vol II. Carbohydrates. Academic Press, London, pp 291–322Google Scholar
  15. Gardiner JC, Taylor NG, Turner SR (2003) Control of cellulose synthase complex localization in developing xylem. Plant Cell 15:1740–1748CrossRefGoogle Scholar
  16. Geisler-Lee J, Geisler M, Coutinho PM, Segerman B, Nishikubo N, Takahashi J, Aspeborg H, Djerbi S, Master E, Andersson-Gunneras S, Sundberg B, Karpinski S, Teeri TT, Kleczkowski LA, Henrissat B, Mellerowicz EJ (2006) Poplar carbohydrate-active enzymes. Gene identification and expression analyses. Plant Physiol 140:946–962CrossRefGoogle Scholar
  17. Grimsley NH, Ashton NW, Cove DJ (1977) The production of somatic hybrids by protoplast fusion in the moss, Physcomitrella patens. Mole Gen Genet 154:97–100CrossRefGoogle Scholar
  18. Hara K, Morita M, Takahashi R, Sugita M, Kato S, Aoki S (2001) Characterization of two genes, Sig1 and Sig2, encoding distinct plastid ơ factor in the moss Physcomitrella patens: phylogenetic relationships to plastid σ factors in higher plants. FEBS Lett 499:87–91CrossRefGoogle Scholar
  19. Hebant C (1977) The conducting tissues of bryophytes. J Cramer, Vaduz, Liechtenstein, p 157Google Scholar
  20. Hohe A, Rensing SA, Mildner M, Lang D, Reski R (2002) Day length and temperature strongly influence sexual reproduction and expression of a novel MADS-box gene in the moss Physcomitrella patens. Plant Biol 4:595–602CrossRefGoogle Scholar
  21. Holland N, Holland D, Helentjaris T, Dhugga KS, Xoconostle-Cazares B, Delmer DP (2000) A comparative analysis of the plant cellulose synthase (CesA) gene family. Plant Physiol 123:1313–1323CrossRefGoogle Scholar
  22. Kamisugi Y, Schlink K, Rensing SA, Schween G, Stacklberg MV, Cuming AC, Reisk R, Cove D (2006) The mechanism of gene targeting in Physcomitrella patens: homologous recombination, concatenation and multiple integration. Nucleic Acids Res 34:6205–6214CrossRefGoogle Scholar
  23. Kimura S, Laosinchai W, Itoh T, Cui XJ, Linder CR, Brown RM (1999) Immunogold labeling of rosette terminal cellulose-synthesizing complexes in the vascular plant Vigna angularis. Plant Cell 11:2075–2085CrossRefGoogle Scholar
  24. Knight CD, Cove DJ, Cuming AC, Quatrano RS (2002) Moss gene technology. In: Gilmartin PM, Bowler C (eds) Molecular plant biology, vol 2. Oxford University Press, Oxford, pp 285–301Google Scholar
  25. Kurek I, Kawagoe Y, Jacob-Wilk D, Doblin M, Delmer D (2002) Dimerization of cotton fiber cellulose synthase catalytic subunits occurs via oxidation of the zinc-binding domains. Proc Natl Acad Sci USA 99:11109–11114CrossRefGoogle Scholar
  26. Muller SC, Brown RM Jr (1980) Evidence for an intramembranous component associated with a cellulose microfibril synthesizing complex in higher plants. J Cell Biol 84:315–316CrossRefGoogle Scholar
  27. Nishiyama T, Hiwatashi Y, Sakakibara K, Kato M, Hasebe M (2000) Tagged mutagenesis and gene-trap in the moss, Physcomitrella patens by shuttle mutagenesis. DNA Res 7:1–9CrossRefGoogle Scholar
  28. Pear JR, Kawagoe Y, Schreckengost WE, Delmer DP, Stalker DM (1996) Higher plants contain homologs of the bacterial celA genes encoding the catalytic sub-unit of cellulose synthase. Proc Natl Acad Sci USA 93:12637–12642CrossRefGoogle Scholar
  29. Persson S, Paredez A, Carroll A, Palsdottir H, Doblin M, Poindexter P, Khitrov N, Auer M, Somerville CR (2007) Genetic evidence for three unique components in primary cell-wall cellulose synthase complexes in Arabidopsis. Proc Natl Acad Sci USA 104:15566–15571CrossRefGoogle Scholar
  30. Rensing SA, Ick J, Fawcett JA, Lang D, Zimmer A, Van de Peer Y, Reski R (2007) An ancient genome duplication contributed to the abundance of metabolic genes in the moss Physcomitrella patens. BMC Evol Biol 7:130CrossRefGoogle Scholar
  31. Richmond T (2000) Higher plant cellulose synthases. Genome Biol 4:30011–30016Google Scholar
  32. Richmond TA, Somerville CR (2000) The cellulose synthase superfamily. Plant Physiol 124:495–498CrossRefGoogle Scholar
  33. Roberts A, Bushoven JT (2007) The cellulose synthase (CesA) gene superfamily of the moss Physcomitrella patens. Plant Mol Biol 63:207–219CrossRefGoogle Scholar
  34. Roberts AW, Roberts E (2004) Cellulose synthase (CesA) genes in algae and seedless plants. Cellulose 11:419–435CrossRefGoogle Scholar
  35. Saxena IM, Lin FC, Brown RM Jr (1990) Cloning and sequencing of the cellulose synthase catalytic sub-unit gene of Acetobacter xylium. Plant Mol Biol 15:673–683CrossRefGoogle Scholar
  36. Saxena IM, Brown RM Jr, Dandekar T (2001) Structure-function characterization of cellulose synthase: relationship to other glycosyltransferases. Phytochemistry 57:1135–1148CrossRefGoogle Scholar
  37. Schaefer DG (2002) A new moss genetics: targeted mutagenesis in Physcomitrella patens. Annu Rev Plant Biol 53:477–501CrossRefGoogle Scholar
  38. Schaefer DG, Zryd JP (1997) Efficient gene targeting in the moss Physcomitrella patens. Plant J 11:1195–1206CrossRefGoogle Scholar
  39. Schaefer DG, Zryd JP (2001) The moss Physcomitrella patens, now and then. Plant Physiol 127:1430–1438CrossRefGoogle Scholar
  40. Schaefer D, Zryd JP, Knight C, Cove DJ (1991) Stable transformation of the moss Physcomitrella patens. Mol Gen Genet 226:418–424Google Scholar
  41. Smith GM (1955) Cryptogamic botany, vol II. Bryophytes and pteridophytes, 2nd edn. McGraw-Hill, New YorkGoogle Scholar
  42. Somerville C (2006) Cellulose synthesis in higher plants. Annu Rev Cell Dev Biol 22:53–78CrossRefGoogle Scholar
  43. Tanaka K, Murata K, Yamazaki M, Onosato K, Miyao A, Hirochika H (2003) Three distinct rice cellulose synthase catalytic subunit genes required for cellulose synthesis in the secondary wall. Plant Physiol 133:73–83CrossRefGoogle Scholar
  44. Taylor NG (2008) Cellulose biosynthesis and deposition in higher plants. New Phyto 178:239–252CrossRefGoogle Scholar
  45. Taylor NG, Laurie S, Turner SR (2000) Multiple cellulose synthase catalytic subunits are required for cellulose synthesis in Arabidopsis. Plant Cell 12:2529–2540CrossRefGoogle Scholar
  46. Taylor NG, Howells RM, Huttly AK, Vickers K, Turner SR (2003) Interactions among three distinct CesA proteins essential for cellulose synthesis. Proc Natl Acad Sci USA 100:1450–1455CrossRefGoogle Scholar
  47. Tseko I (1999) The sites of cellulose synthesis in algae: diversity and evolution of cellulose-synthesizing enzyme complexes. J Phycol 35:635–655CrossRefGoogle Scholar
  48. Tusnády GE, Simon I (1998) Principles governing amino acid composition of integral membrane proteins: applications to topology prediction. J Mol Biol 283:489–506CrossRefGoogle Scholar
  49. Tusnády GE, Simon I (2001) The HMMTOP transmembrane topology prediction server. Bioinformatics 17:849–850CrossRefGoogle Scholar
  50. Wong HC, Fear AL, Calhoon RD, Eichinger GH, Mayer R, Amikam D, Benziman M, Gelfand DH, Meade JH, Emerick AW et al (1990) Genetic organization of the cellulose synthase operon in Acetobacter xylinum. Proc Natl Acad Sci USA 87:8130–8134CrossRefGoogle Scholar
  51. Zhang JZ (2003) Evolution by gene duplication: an update. Trends Ecol Evol 18:292–298CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Hua Zhang Wise
    • 1
  • Inder M. Saxena
    • 1
  • R. Malcolm BrownJr.
    • 1
    Email author
  1. 1.Section of Molecular Genetics and MicrobiologyThe University of Texas at AustinAustinUSA

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