TITLE: Hormone-mediated regulation of cellular development in the moss, Physcomitrella patens (EUROMOSS)


Start date: 1.1.94 Duration: 3 years 4 months

 COORDINATOR: Trevor L Wang , John Innes Centre, Norwich UK NR4 7UH 



The focus of the programme is to isolate auxin- and cytokinin-regulated genes using differential cDNA screening and heterologous probing, determine the interaction with light in their expression and determine which are important in regulating plant morphogenesis. Additional objectives are to improve plant transformation and gene tagging technologies and to develop a plant complementation system.



Gene isolation: task 1 (A4.1, A4.2, A4.5, A4.6, A4.7). Several cDNA libraries ( gt10 and ZAP) have been constructed and screened differentially. These are from different stages of the wild type (WT) and from tissue treated with cytokinin for different times. Four cytokinin-modulated cDNAs have been sequenced, two show homology to ribosomal proteins, one has no homologues on the databases and one is similar to differentiation specific genes. One other cDNA (321) is expressed in the wild type, but not in a cytokinin-responsive mutant. Further characterisation of these cDNAs including the use of reverse genetics is being carried out by A4.1, A4.6. As a prelude to studies on auxin-regulated genes, new auxin resistant mutants have been isolated including mutants (cT83, sT33) deficient in gametophores that can be rescued by auxin. Homeotic genes (moss flo) homologous to the Antirrhinum floricaula gene have been cloned. These genes are not activated until 24 hours after cytokinin treatment once gametophore development is fixed. Two purine metabolising enzymes - adenosine phosphoribosyl transferase (APRT) and adenosine kinase (AK) - have been cloned (A4.2) in collaboration with A4.6 by complementation of E.coli mutants and genomic clones isolated (A4.1). Several plastid proteins have been shown to be regulated by cytokinin. They have been identified by microsequencing as OEE2, PGK and subunit of chloroplast ATPase (A4.6). Complementation studies are continuing using new and improved vectors (A4.4). Random sequencing or heterologous probing has been used to isolate a number of other genes including pyruvate kinase, a histone, a moss homologue of Drosophila pumilio and cell cyle genes (A4.4). The functions of all these genes are being examined using reverse genetics including gene disruption.

Hormone binding proteins and genes: task 2 (A4.2, A4.5). Attempts to obtain P. patens homologues of maize and Arabidopsis thaliana auxin binding proteins (ERabp1) by PCR using degenerate oligos (A4.5) did not prove successful. Preliminary attempts to find cytokinin-binding activity in moss chloroplasts (a potential target organelle for cytokinin) using tritiated azido-CCPU (a cytokinin analogue) have not detected any binding proteins (A4.2). Similar labelling studies with cytosolic fractions has detected a 34kDa peptide that has been purified to near-homogeneity by affinity chromatography and a number of peptides partially sequenced following digestion by endolysine C. One peptide shows an interesting homology with mammalian ARNT (arylhydrocarbon receptor nuclear translocator) proteins.

Cell biology studies: task 3 (A4.3, A4.1). Protocols for LM, SEM and TEM have been established for the investigation of protonemal growth in wild-type and mutant strains. During detailed analyses of the wild type, differences in morphogenesis have been observed that depend on the type of inoculum employed. In addition, ca. 17 mutant strains and a number of transgenics have been examined and compared to the wild type in collaboration with A4.1, A4.4, A4.5, A4.6. Whole mount in situ mRNA hybridization using digoxygenin-labelled cRNAs has been attempted, but initial results were misleading and probe penetration has proved difficult. New protocols are currently being investigated (A4.1, A4.3).

Metabolic studies: task 4 (A4.2). Preliminary evidence obtained using tritiated adenine and isopentenyladenosine has indicated that movement of cytokinins and metabolites differs between wild type and ove mutants. Functional complementation of E. coli mutants enabled the isolation of genes encoding an adenine phosphoribosyltransferase (APRT) and an adenosine kinase, which catalyse the formation of AMP from adenine and adenosine respectively. The cDNAs were expressed in E.coli and enzyme activities investigated. The data obtained indicated that AK is the only pathway responsible for cytokinin nucleotide formation in Physcomitrella, since iP is not converted to isopentenyladenosine-5'-monophosphate by APRT in vitro and in vivo, in contrast to what is observed in higher plants.

Transformation studies, reverse genetics and gene disruption: task 4 (A4.1, A4.4, A4.6, A4.7). Transformation using PEG, explosive and electrostatic microprojectile techniques have all been used in moss. Evidence for homologous recombination and gene silencing during integration have been obtained. Gene targeting experiments have been attempted and proved successful, with an 80% efficiency for the 3 genomic loci and 6 artificial loci examined (A4.7). Disruption of genes encoding APRT and chlorophyll a/b binding protein has been achieved with direct replacement of APRT using a cDNA replacement vector producing plants with the correct phenotype (diaminopurine resistance). Constructs for antisense work have been made with either mono- and bidirectional promoters e.g. pLUG3 (A4.4). Transformation frequencies between 2 and 175 per µg plasmid DNA have been achieved. The function of the moss flo genes and several cytokinin-modulated genes have been examined by transformation in sense and antisense orientation. The phenotypes indicate they are involved in leafy shoot development. Gene disruption has been started with the moss flo genes. Several stable ipt transformants with the expected phenotype (bud overproduction) have been made and their physiology and cell biology has been studied (A4.6, A4.3 and Halle).



1. The first demonstration of gene disruption in plants. Disruption of genes encoding adenosine phosphoribosyl transferase and cab achieved.
2. Efficient gene targeting achieved.
3. Isolation of cDNAs and genomic clones encoding the purine metabolising enzymes, adenosine phosphoribosyl transferase and adenosine kinase using complementation of E. coli mutants.
4. Isolation of cDNAs and genomic clones for homeotic, floricaula-like genes and demonstration using trangenics of involvement in leafy shoot formation.
5. Isolation of cDNAs and genomic clones for moss phytochrome.
6. Isolation of 4 cytokinin-modulated cDNAs, two encoding ribosomal proteins, and demonstration using transgenics that one is involved in leafy shoot formation.
7. Evidence for homologous recombination and gene silencing in stable transformants.
8. Demonstration of cytokinin-regulation of three plastid polypeptides, OEE2, PGK and subunit of ATPase.
9. Partial sequence obtained for a 34kDa cytokinin-binding protein (Pp34) isolated by photoaffinity labelling.



Total: 8 Joint: 1

Hadeler, B., Scholz, S., Reski, R. (1995). Gelrite and agar differently influence cytokinin-sensitivity of a moss. J. Plant Physiology 146, 369-71.
Leech, M.J., Russell, A.J. and Wang, T.L. Probing cytokinin action II. Abstract no. 305. 15th Intl. Conf. Plant Growth Substances, Minneapolis, 1995.
Reski, R., K. Reutter, B. Kasten, M. Faust, S. Kruse, G. Gorr, R. Strepp, W.O. Abel (1995) In: Current Issues in Plant Molecular and Cellular Biology (Terzi, M., Cella, R., Falavigna, eds.), pp.291-296. Kluwer, Dordrecht.
Hofmann A., Simeone A., Gargiulo M., Tortora M., Cafiero G., Gambardella R., Russo VEA. 1996. Morphological analysis of the life cycle of the moss Physcomitrella patens. Botanica Acta (submitted).
Kasten, B., F. Buck, G. Gorr, J. Nuske, R. Reski (1996). Cytokinin interacts with the photoreceptors and affects nuclear- as well as plastome-encoded energy-converting plastid enzymes. The Plant Journal (submitted).
Kasten, B. and Reski, R. (1996): -lactam antibiotics inhibit chloroplast division in a moss (Physcomitrella patens) but not in tomato (Lycopersicon esculentum). J Plant Physiol. (submitted).
Reutter, K., R. Atzorn, T. Schmülling, R. Strepp, R. Reski (1996): Expression of the bacterial ipt-gene cures the mutation in plastid division but not the mutation in gametophore development in a cytokinin-sensitive moss mutant. The Plant Journal, submitted.
Schaefer, D. and Zryd, J.-P. Efficient gene targeting in the moss Physcomitrella patens, Plant Journal (submitted).



Antisense, auxin, cytokinin, floricaula, gene disruption, gene expression, gene targeting, homeotic genes, metabolism, morphogenesis, moss, Physcomitrella patens, purine, ribosomal protein, transformation



Trevor Wang (A4.1; Coordinator)

John Innes Centre

GB - Norwich

Raffaele Gambardella (A4.3)

University of Naples

I - Naples

Enzo Russo (A4.5)

MPI Moleculare Genetik

D - Berlin

Jean-Pierre Zrÿd (A4.7)

University of Lausanne

CH - Lausanne

Michel Laloue (A4.2)


F - Versailles

David Cove (A4.4)

University of Leeds

GB - Leeds

Ralf Reski (A4.6)

University of Hamburg

D - Hamburg



 Progress on milestones





First 12 months

Task 1: Initiate search via differential cDNA library screening for auxin and cytokinin-regulated genes; probe libraries for hormone-regulated genes from other species; initiate studies on mapping, complementation and transposon tagging.

Libraries have been constructed and screened. Heterologous probing started as well as other studies on mapping etc.

4.1, 4.4, 4.5, 4.6

Task 2: Initiate library screening for genes involved in hormone and light-signalling using probes from other species and by differential screening of libraries from dark-grown and light-induced tissues. Initiate isolation of binding proteins for auxin and cytokinin using affinity probes.

Heterologous probing for hormone genes started. Azido affinity probe binding studies started for cytokinin.

4.2, 4.5, 4.7

Task 3: Initiate light and electron microscopy studies on moss morphology in wild type and mutants. Initiate immunofluorescence studies using anti-cytoskeletal antibodies.

Microscopy studies started on wild type and selected mutants.

4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7

Task 4: Carry out studies on auxin and cytokinin metabolism in wild type and mutant mosses.

Cytokinin metabolic studies started on wild type and ove mutants.

4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7

Second 12 months

Task 1: Complete differential screening and heterologous probing. Continue studies on mapping, complementation and transposon tagging. Initiate genomic library screening.

Screening completed, cytokinin-modulated cDNAs isolated, developmentally-regulated cDNAs isolated and genomics obtained. cDNAs encoding two purine-metabolising enzymes (APRT and AK) isolated. Initial heterologous probing unsuccessful for auxin genes. Complementation and tagging held in abeyance while gene targeting attempted.

4.1, 4.4, 4.6, 4.7

Task 2: Complete studies using affinity probes.

Cytokinin-binding protein isolated and purified.

4.2, 4.4

Task 3: Initiate studies on gene expression using in situ mRNA hybridisation with cDNAs from first year.

In situ studies initiated, but proving difficult.

4.1, 4.3

Task 4: Initiate production of constructs for transformation and production of transgenics using cDNAs isolated in first year. Initiate studies on hormone metabolism in transgenics.


Additional work carried out: attempt to disrupt genes isolated in previous years by targeting individual loci


Constructs made and transgenics for moss flo, ipt and cytokinin-modulated cDNAs made. ipt transgenics analyzed for cytokinin metabolism.



Constructs made for replacement and insertion vectors for known sequences

A4.1, 4.2, 4.4, 4.6,





4.2, 4.4, 4.5, 4.7

Third 12 months 

Task 1: Complete isolation of genomic clones. Continue mapping, complementation and tagging studies.

Isolation of genomics still in progress, complementation and tagging reinitiated using new constructs. Phytochrome gene isolated as well as several other randomly isolated cDNAs.

4.1, 4.4, 4.6

Task 2: Library screening for genomic clones and cDNAs corresponding to affinity isolated binding proteins.

Continuing. New round of heterologous probing started for auxin genes.

4.1, 4.2, 4.6

Task 3: Study the morphological characteristics of transgenics. Complete in situ hybridisation studies and extend both in situ and immunofluorescent studies to transgenics.

Morphological analysis of transgenic flo and ipt plants started. New in situ protocols for whole mount work being examined.

4.1, 4.3

Task 4: Continue production of constructs for transformation studies and production of transgenics using genes isolated in previous years. Analyse hormone metabolism in transgenics.


Additional work carried out: attempt to disrupt genes APRT and cab genes isolated in previous years by targeting


Work continues. New constructs being made for developmentally-regulated cDNAs. Transgenic analysis continuing.



Targeting of known sequences achieved and disruption of APRT and cab genes achieved.

4.1, 4.2, 4.4, 4.5, 4.6, 4.7



4.4, 4.5,




Theme A4 EUROMOSS: press release


'Target practice with moss'

 Almost from the birth of the science of genetics, mosses have been recognised as outstanding research tools and, by 1940, they had been established the organisms of choice for genetic studies. Moss research has kept apace of new technologies and, in recent years, it has adopted molecular biology with relish. Although existing largely in the shadow of research on higher plants since then, it is now on the verge of overtaking the rest of plant biology through the recent discovery that gene targeting is feasible on a routine basis in moss. Targeting permits the possibility of disrupting the activity of genes so that we can find out what they do. The potential consequences for plant research are enormous.

Questions, remarks, suggestions? Please contact Micha Chakhparonian