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