Ralf Reski
Freiburg University, Plant Biotechnology, Sonnenstr. 5, D-79104 Freiburg, Germany (email: ralf.reski@biologie.uni-freiburg.de)
Key words EST, gene targeting, homologous recombination, moss
The post-genomic era relies on a variety of high-throughput approaches for the establishment of stringent gene/function correlations (e.g. Holtorf et al.
2002a). One of the most powerful tools is reverse genetics, where the targeted disruption of a specific ORF leads to a loss-of-function mutation, which in turn establishes the biological role of this specific gene. Unlike all other land plants analyzed so far, the moss Physcomitrella patens exhibits a very high rate of homologous recombination in its nuclear DNA, making gene targeting approaches in this plant as convenient as in yeast or in ES cells of mice (e.g. Reski 1998, 1999). We, therefore, have developed
Physcomitrella as a novel tool in plant functional genomics.
In mosses the dominating generation is the haploid gametophyte, which facilitates analysis of loss-of-function mutations without complex backcrosses. Futhermore, several tissues are only one cell-layer thick, making them unique targets for advanced cell biological studies. Thus, we have identified the first organelle dividing protein in any eukaryote (Strepp et al.
1998), provided evidence for a plastoskeleton (Kiessling et al. 2000, Reski
2002), and identified a novel mechanism for dual, but distinct targeting of nuclear-encoded proteins into mitochondria and plastids (Richter et al.
2002). In order to better understand the role of cell walls during plant growth, we expansins were analyzed by targeted knock-out (Schipper et al.
2002). In order to study embryo development in Physcomitrella we
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developed a highly standadized protocol for the induction of sexual reproduction in this species (Hohe et al. 2002a).
Physcomitrella is far more robust against abiotic stresses like drought and salt than many seed plants including Arabidopsis, making the moss a valuable source for the identification of novel resistance genes (Frank and
Reski 2002). Moreover, Physcomitrella is rich in secondary metabolites and in very long and unsaturated fatty acids promoting human health.
Subsequently, a variety of novel genes like elongases and desaturases were identified (Girke et al. 1998, Zank et al. 2002, Sperling et al. 2002).
Likewise, functional knockout of the apr gene revived an old route of sulfate assimilation in plants (Koprivova et al. 2002).
Most transformation protocols of Physcomitrella rely on direct DNA transfer to protoplasts. For large-scale plant production a bioreactor regime was established that allows a tight control of growth and differentiation (Hohe et al. 2002b) which was subsequently optimized for mass production of protoplasts (Hohe and Reski 2002). Protoplast regeneration was studied in detail (Bhatla et al. 2002) and protocols were optimized for efficient regeneration (Schween et al. 2002a). Furthermore, the effects of different parameters on transformation as well as on gene targeting were elucidated
(Hohe et al. 2002c), leading to very efficient gene targeting (Egener et al.
2002a).
The Physcomitrella genome is about three times as big as the Arabidopsis genome and is distributed on 27 chromosomes (Reski et al. 1994). Small
EST-sequencing efforts (Reski et al. 1998, Machuka et al. 1999) revealed that Physcomitrella is a rich source of novel genes although the majority of genes are significantly conserved between moss and seed plants. In a more thorough approach normalized and subtracted cDNA collections covering all steps of the life cycle have been sequenced. About 110,000 ESTs were generated and from that a clustered database was constructed covering more than 95% of the transcriptome (~ 25,000 genes) matching ~ 50% of the ~
26,000 Arabidopsis genes (Rensing et al. 2002).
As a prerequisite for a large-scale gene/function correlation study, we are establishing a collection of transgenics with insertion mutations in most expressed genes. Low-redundancy moss cDNA libraries are mutagenized in
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E. coli and used for plant transformation. These disruption constructs are expected to target preferentially expressed genes. Among the first over
20,000 transgenics ~ 16% deviated from wild-type in a variety of developmental, morphological and physiological characteristics (Egener et al. 2002b), exceeding by far the respective figures from similar Arabidopsis approaches. Our constant production capacity is 160 transgenics per day,
800 per week. For optimization different promoters were tested (Holtorf et al. 2002b), and efficient protocols for DNA isolation (Schlink and Reski
2002) and high-throughput PCR were developed (Schween et al. 2002b).
Thus, Physcomitrella has been established as a novel tool in plant functional genomics, utilizing the unique moss features like homologous recombination and a variety of genes so far not identified in higher plants. Details can be found under www.plant-biotech.net and we are open for the establishment of novel co-operations.
Acknowledgements
This work was made possible by a variety of dedicated colleagues and coworkers, all cited in the references. Financial support came from BPS, DFG,
A.-v.-H.-Stiftung, FCI, MWK and Freiburg University.
Bhatla S.C., J. Kiessling, and R. Reski. 2002. Observation of polarity induction by cytochemical localization of phenylalkylamine-binding receptors in regenerating protoplasts of the moss Physcomitrella patens . Protoplasma 219:99-105.
Egener T., A. Hohe, J.M. Lucht, and R. Reski. 2002a. High throughput gene targeting by protoplast transformation of Physcomitrella patens . Submitted.
Egener T., J. Granado, M.-C. Guitton, A. Hohe, H. Holtorf, J.M. Lucht, S. Rensing, K.
Schlink, J. Schulte, G. Schween, S. Zimmermann, E. Duwenig, B. Rak, and R. Reski. 2002b.
High frequency of phenotypic deviations in Physcomitrella patens plants transformed with a gene-disruption library. Submitted.
Frank W., and R. Reski. 2002. Physcomitrella patens is highly tolerant against drought, salt and osmotic stress. Submitted.
Girke T., H. Schmidt, U. Zähringer, R. Reski, and E. Heinz. 1998. Identification of a novel
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6-acyl-group desaturase by targeted gene disruption in Physcomitrella patens.
Plant J.
15:39-48.
Hohe A., and R. Reski. 2002. Optimisation of a bioreactor culture of the moss Physcomitrella patens for mass production of protoplasts. Plant Sci., in press.
Hohe A., S.A. Rensing, M. Mildner, D. Lang, and R. Reski. 2002a. Day length and temperature strongly influence sexual reproduction and expression of a novel MADS-box gene in the moss Physcomitrella patens.
Plant Biology, in press.
Hohe A., E.L. Decker, G. Gorr, G. Schween, and R. Reski. 2002b. Tight control of growth and cell differentiation in photoautotrophically growing moss ( Physcomitrella patens ) bioreactor cultures. Plant Cell Rep., in press.
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Hohe A., T. Egener, J. Lucht, H. Holtorf, C. Reinhard, G. Schween, and R. Reski. 2002c.
DNA conformation and plant pre-culture influence both, transformation efficiency and gene targeting efficiency in Physcomitrella. Submitted.
Holtorf H., M.-C. Guitton, and R. Reski. 2002a. Plant functional genomics.
Naturwissenschaften, online-publication available.
Holtorf H., A. Hohe, H.-L. Wang, M. Jugold, T. Rausch, E. Duwenig, and R. Reski. 2002b.
Promoter subfragments of the sugar beet V-type H + -ATPase subunit c isoform drive the expression of transgenes in the moss Physcomitrella patens . Submitted.
Kiessling J., S. Kruse, S.A. Rensing, K. Harter, E.L. Decker, and R. Reski. 2000.
Visualization of a cytoskeleton-like FtsZ network in chloroplasts. J. Cell Biol. 151:945-950.
Koprivova A., A.J. Meyer, G. Schween, C. Herschbach, R. Reski, and S. Kopriva. 2002.
Functional knockout of the adenosine 5´phosphosulfate reductase gene in Physcomitrella patens revives an old route of sulfate assimilation. Submitted.
Machuka J., S. Bashiardes, E. Ruben, K. Spooner, A. Cuming, C. Knight, and D. Cove. 1999.
Sequence analysis of expressed sequence tags from an ABA-treated cDNA library identifies stress response genes in the moss Physcomitrella patens . Plant Cell Physiol 40:378-387.
Rensing S.A., S. Rombauts, A. Hohe, D. Lang, E. Duwenig, P. Rouze, Y. Van de Peer, and R.
Reski. 2002. The transcriptome of the moss Physcomitrella patens : comparative analysis reveals a rich source of new genes. Submitted.
Reski R. 1998. Physcomitrella and Arabidopsis: the David and Goliath of reverse genetics.
Trends Plant Sci 3:209-210.
Reski R. 1999. Molecular genetics of Physcomitrella. Planta 208:301-309.
Reski R. 2002. Rings and networks: the amazing complexity of FtsZ in chloroplasts. Trends
Plant Sci. 7:103-105.
Reski R., M. Faust, X.-H. Wang, M. Wehe, and W.O. Abel. 1994. Genome analysis of the moss Physcomitrella patens (Hedw.) B.S.G. Mol. Gen. Genet. 244:352-359.
Reski R., S. Reynolds, M. Wehe, T. Kleber-Janke, and S. Kruse. 1998. Moss ( Physcomitrella patens ) expressed sequence tags include several sequences which are novel for plants. Bot.
Acta 111:143-149.
Richter U., J. Kiessling, B. Hedtke, E. Decker, R. Reski, T. Börner, and A. Weihe. 2002.
Phage-type RNA polymerases of Physcomitrella patens : two enzymes with dual targeting to mitochondria and plastids. Gene, in press.
Schipper O., D. Schaefer, R. Reski, and A. Fleming. 2002. Expansins in the bryophyte
Physcomitrella patens . Plant Mol. Biol., in press.
Schlink K., and R. Reski. 2002. Reliable method for preparation of high-quality DNA from the moss Physcomitrella patens.
Submitted.
Schween G., A. Hohe, A. Koprivova, and R. Reski. 2002a. Effects of nutrients, cell density and culture techniques on protoplast regeneration and early protonema development in
Physcomitrella patens.
Submitted.
Schween G., S. Fleig, and R. Reski. 2002b. High-throughput-PCR screen of 15,000 transgenic
Physcomitrella plants. Plant Mol. Biol. Rep. 20:43-47.
Sperling P., T. Egener, J. Lucht, R. Reski, P. Cirpus, and E. Heinz. 2002. Identification of a
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5-fatty acid desaturase from Physcomitrella patens.
Adv. Res. Plant Lipids, in press.
Strepp R., S. Scholz, S. Kruse, V. Speth, and R. Reski. 1998. Plant nuclear gene knockout reveals a role in plastid division for the homolog of the bacterial cell division protein FtsZ, an ancestral tubulin. Proc. Natl. Acad. Sci. USA 95:4368-4373.
Zank T.K., U. Zähringer, C. Beckmann, G. Pohnert, W. Boland, H. Holtorf, R. Reski, J.
Lerchl, and E. Heinz. 2002. Cloning and functional characterisation of an enzyme involved in the elongation of Δ6-polyunsaturated fatty acids from the moss
Physcomitrella patens.
Plant
J., in press.
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