Progress 07/01/03 to 06/30/06
Outputs
The complete CESA gene superfamily of the moss Physcomitrella patens was analyzed by screening cDNA and genomic libraries, identifying and sequencing full-length cDNA sequences produced by the Physcobase (http://moss.nibb.ac.jp/) and PEP (University of Leeds) projects and by searching of the complete genome sequence produced by the Joint Genome Institute (http://www.jgi.doe.gov/index.html) before and after assembly. This analysis lead to the following insights: 1) Three of the seven families that compose the CESA superfamilies of Arabidopsis and Populus trichocarpa are absent from the P. patens genome. These include the CSLB, CSLE and CSLG families, for which the biological functions are unknown. The CSLF and CSLH families, found in rice, but not Arabidopsis and P. trichocarpa, are also absent from the P. patens genome. 2) The gene families that are shared among P. patens and seed plants diversified independently in the moss lineage. Thus, P.patens does not have
orthologs of the seed plant CESAs whose products are thought to have distinct roles in the assembly and function of cellulose-synthesizing rosette terminal complexes. 3) Members of the CSLD gene family are more highly expressed in P. patens than in any of the seed plants. This is consistent with the proposed function of CSLD genes in tip growth of pollen tubes and root hairs and the central role of tip growth in the development of the moss protonema. A total of 30 CESA and CSL gene have been manually-curated in the P. patens genome. These insights lead to a shift in our approach to using P. patens for functional analysis of CESA and CSL genes. The original plan to investigate terminal complex assembly using CESA knockouts was deemed unlikely to reveal the assembly mechanism for heteromeric seed plant terminal complexes, since P. patens lacks orthologs of the individual seed plant CESA monomers. However, the absence of CSLB, CSLE, CSLG and CSLH genes suggested that P. patens could be
used as a heterologous expression system to investigate the functions of these gene families. Expression vectors containing the coding sequences of Arabidopsis CSLB3, CSLE1 and CSLG1 genes were constructed and used to transform P. patens. Genetic analysis of stable transformants is now underway. In collaboration with William Willats (University of Copenhagen), a rapid immunomicroarray screen for changes in P. patens cell wall polysaccharide content has been developed (manuscript in preparation) and will be used to analyze heterologous expression lines. In collaboration with Aaron Liepman (Michigan State University), P. patens CSLA coding sequences were expressed in insect cells. The expressed proteins have mannan and glucomannan synthase activity, demonstrating conservation of function among seed plant and moss CSLA genes (manuscript in preparation). CSLC and CSLD genes are also being knocked out to investigate their functions in xyloglucan biosynthesis and tip growth, respectively.
TAIL PCR was used to complete the sequence of a CESA gene from the red alga Porphyra yezoensis. A partial sequence of a CSLD gene from the green alga Coleochaete scutata was also cloned.
Impacts
Unanticipated results of this project indicate that heterologous expression in P. patens provides a unique approach to investigating the functions of previously uncharacterized enzymes involved in assembly of the plant cell wall. A better understanding of plant cell wall assembly will enable modification of the raw materials used to manufacture textiles, paper, packaging, building materials, and many other products. This knowledge may also contribute to human health through improvement of dietary fiber and to environmental protection through enhanced production of biomass fuels.
Publications
- Roberts, A. W., Bushoven, J.T. 2006. The cellulose synthase (CESA) gene superfamily of the moss Physcomitrella patens. Plant Molecular Biology (DOI 10.1007s11103-9083-1)
- Roberts, A. W., Roberts, E. M. 2004. Cellulose synthase (CesA) genes in algae and seedless plants. Cellulose 11:419-435
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Progress 10/01/04 to 09/30/05
Outputs
Characterization of the CesA/CslD families in Physcomitrella patens (Objective 1): Through similarity searches using vascular plant CesA and CslD genes as queries against the Physcobase P. patens EST database, full-length cDNA clones from seven CesA genes, eight CslD genes, three CslC genes and two CslA genes from P. patens were identified and sequenced. The release of shotgun sequences by the JGI Community Genome Sequencing project for P. patens (5.4 X 109 base pairs, 10-fold coverage) enabled assembly of full length genomic sequences for these genes and also identification of one CesA gene, two CslA genes, three CslC genes, and several pseudogenes that were not represented in the EST database. Intron insertion sites are highly conserved between Arabidopsis and P. patens in all four shared gene families. When Arabidopsis CslBs, CslEs or CslGs or rice CslFs or CslHs were used as queries against both the EST and genomic shotgun databases, no additional CesA superfamily
members were identified in P. patens, indicating that this moss lacks representatives of these families. Phylogenetic analysis strongly supports independent diversification of the shared families in mosses and vascular plants. The lack of orthologs of vascular plant CesAs in the P. patens genome indicates that divergence and specialization of CesAs for primary and secondary cell wall synthesis occurred after the divergence of mosses and vascular plants and that divergence of genes encoding CesA heterotrimers was not necessary for evolution of rosette terminal complexes. In contrast to Arabidopsis, the CslD family is highly represented among P. patens ESTs. This is consistent with the proposed function of CslDs in tip growth and the central role of tip growth in the development of the moss protonema. A manuscript describing the CesA superfamily in P. patens is currently being revised following submission to Plant Physiology. Knockout CesA gene in Physcomitrella (objective 2): We have
designed and constructed replacement vectors for knockout of PpCesA5 and PpCesA8 and are now in the process of using these vectors to transform P. patens. Initial difficulties achieving high rates of protoplast viability have been overcome and antibiotic resistant strains are now in the final round of selection. These strains will be analyzed for targeted integration of the transgenes by PCR using primers within the insert and flanking the integration site. Those that appear to contain the transgene properly integrated at both the 5'and 3' ends will be subjected to Southern analysis to verify the absence of the targeted sequence and Northern analysis or RT-PCR to monitor expression of the transgene. Phenotypes of the knockout line will then be evaluated. Characterization of CesA genes from algae (objective 3): TAIL PCR is being used to obtain full-length clones of a CslD gene from the green alga Coleochaete scutata and a CesA gene from the red alga Porphyra yezoensis.
Impacts
The availability of genomic resources necessary to completely characterize the CesA superfamily in P. patens, which could not have been anticipated when the proposal for this grant was submitted, is an important step toward the development of this organism as a comprehensive model for investigating plant cell wall biosynthesis. For example, the absence of genes representing the CslB, CslE, CslG, CslF and CslH families in P. patens affords an opportunity to investigate the biochemical functions of these genes using a knockin approach.
Publications
- No publications reported this period
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Progress 10/01/03 to 09/30/04
Outputs
Characterization of the Physcomitrella CesA/CslD family (Objective 1): Three genomic and 80 cDNA clones were identified by screening genomic, BAC and cDNA libraries and searching EST collections in one private and two public databases. Genomic clones and 10 full length (about 4.5 Kb each) and 8 partial cDNA clones have been sequenced. Together with the ESTs, these assemble into 15 contigs, 8 similar to seed plant CesA genes and 7 similar to seed plant CslD genes. Three additional full-length CesA cDNA clones have been ordered and will be sequenced. Full-length sequences for the 2 remaining CesA genes will be obtained by 5' RACE. Phylogenetic analysis of full length sequences showed that Physcomitrella CesAs and CslDs cluster together in distinct clades, in marked contrast to seed plant CesAs and CslDs, which form clades consisting of orthologs from different species. Electronic Northern analysis suggests strong differential expression of some genes in protonemata and
leafy gametophytes represented in the different types of cultures from which the ESTs were derived. In contrast to seed plant EST collections, CslDs are over represented compared to CesAs among Physcomitrella ESTs. CslD expression also dominates CesA expression in pollen, which is homologous with moss protonema (both are haploid gametophytes). This is consistent with the hypothesis that CslDs encode the cellulose synthases expressed in tip-growing cells. For detailed expression analysis, we have designed and verified the specificity of primers and used them for preliminary conventional and real time amplification of CesA and CslD cDNA derived from Physcomitrella protonemal cultures. Preliminary in situ RT-PCR experiments have been conducted to develop methods for expression analysis at the tissue and cellular levels. CesA knockout in Physcomitrella (objective 2): PpCesA5 is represented by a complete cDNA sequence, 3 additional cDNA clones, and a nearly-complete genomic sequence, and
is very similar to pseudogene PpCesA2. A 2 Kb fragment of the genomic sequence was used to construct a replacement vector interrupted by a selection cassette. Flanking sequence primers for analysis of transformants were designed to distinguish between targeting of PpCesA5 and pseudogene PpCesA2. If deletion of PpCesA5 is lethal, we predict that all stable transformants will be targeted to the pseudogene. Homologous replacement of either gene will demonstrate the feasibility of targeted transformation. We proposed to send the vector to University of Leeds to be transformed into Physcomitrella, but his service is no longer available. We have regenerated Physcomitrella from protoplasts in preparation for PEG-mediated tranformation. Characterization of CesA genes from algae (objective 3): Using degenerate primers, we have amplified fragments of Coleochaete CesAs. We have isolated genomic DNA and are using the same approach to amplify CesA fragments from Oocystis apiculata and Valonia
ventricosa. We have also obtained an EST clone with high similarity to plant CesAs from the red alga Porphyra yezoensis (Kazusa DNA Research Institute), which will be used as a probe and a basis for designing primers.
Impacts
This work establishes Physcomitrella patens as a model organism for investigating the genetic basis of variation in cellulose microfibril structure. Already known as the only plant in which proficient targeted transformation is possible, Physcomitrella also provides unique opportunities to understand CesA and CslD gene function in seed plants by tracing the evolutionary diversification and functional specialization of this gene family. The large size and unprecedented sequence similarity within the CesA and CslD gene families of Physcomitrella may facilitate identification of functionally-relevant sequence differences, ultimately providing a basis for developing strategies to manipulate microfibril structure in commercial fiber species for enhanced cellulose content and microfibril structure.
Publications
- Roberts, A. W., Roberts, E. M. 2004. Cellulose synthase (CesA) genes in algae and seedless plants. Cellulose 11:419-435
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