Cellulose biosynthesis in Physcomitrella patens
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Physcomitrella patens has become a model system to study plant biology. 8 cellulose synthase (CesA) genes were identified by searching against Physcomitrella EST database. Two of these genes, PpCesA6 and PpCesA7 are the first full-length CesAs to be identified. These two genes are highly similar to each other, both on the cDNA and genomic DNA levels. They both have 13 introns and 12 exons. The first introns are more than 1kb. The proteins they encode both have 1096 amino acids. There are only three amino acid differences in the proteins they encode. PpCesA6 and PpCesA7 share 74% amino acid identity with Monterey pine (Pinus radiate) PrCesA10, 72% amino acid identity with quaking aspen (Populus tremuloide) PtrCesA6, 71% amino acid identity with maize (Zea mays) ZmCesA7 and three rice (Oryza sativa) CesAs, 65%-68% amino acid identity with Arabidopsis CesAs. The deduced proteins of PpCesA6 and PpCesA7 contain the D, D, D, QXXRW motif in the form of DDG, DCD, TED, QVLRW, which is the catalytic region of cellulose synthases. Two other pairs of CesA genes, PpCesA3 and PpCesA8, PpCesA4 and PpCesA10, also show high similarity. PpCesA2 and PpCesA9 are pseudogenes. By taking advantage of the high efficiency homologous recombination in Physcomitrella nuclear DNA, a C-terminus GFP fusion construct was produced for PpCesA6. Expression analysis showed that PpCesA6 is expressed in both protonemata and young gametophore. In protonemata, PpCesA6 is expressed in both chloronema and caulonema cells, but not in every cell. In young gametophore, PpCesA6 is expressed in axillary hairs and rhizoids. Confocal miscrocopy study shows that PpCesA protein is localized on the plasma membrane and it is randomly dispersed. The gene targeted knockout constructs of PpCesA6 and PpCesA7 were produced. The null mutants of PpCesA6 and PpCesA7 single knockout as well as double knockout were generated by the PEG (polyethylene glycol)-mediated protoplast transformation. Both single knockout mutants did not show obvious phenotypic differences from the wild type. The double knockout mutants had reduced stem length. The stem lengths of the wild type, PpCesA6 knockout mutant, PpCesA7 knockout mutant and double knockout mutant growing on BCD and BCDAT media were 3.93±0.45mm and 3.51±0.08mm, 3.82±0.46mm and 3.5±0.3mm, 3.65±0.68mm and 3.73±0.49mm, 2.75±0.22mm and 2.65±0.43mm, respectively. A cellulose synthase-like C gene (CslC4) was identified by searching against the Physcomitrella EST and genomic DNA databases. The protein it encodes is 694 amino acids. The D, D, D, QXXRW motif is in the form of DDS, DAD, VED, QQHRW. PpCslC4 genomic DNA has 4 small introns in the coding region. There is also one small intron at the 5'-UTR. The deduced PpCslC4 protein shows 72% similarity with PpCslC2 and PpCslC3, 65% similarity with PpCslC1. When compared with other organisms, PpCslC4 protein shows more than 60% similarity with Arabidopsis and Oryza sativa CslC proteins. A gene targeted knockout construct was produced for PpCslC4. The null mutants were generated by the PEG-mediated protoplast transformation. PpCslC4 mutant did not show any obvious phenotypic differences from the wild type.