PIP Newsletter #7: Jan. 1995

PIP draws your attention to the following issues:


1. Meetings. The 'Project of technological Priority' Theme B2 and A1 groups have planned research meetings in February and March. PIP members are invited to attend the meeting and to take part in the discussions. The meetings will focus on "Molecular and Cellular Studies of the role of membrane transport processes in plant responses to environment and to abiotic stresses, including salinity and potassium deficiency" and on "Pattern Formation in Plant Embryos; study of the transition from vegetative to reproductive growth and of the control of flowers and seeds, as well as the determination of leaf shape, size and number of axillary shoot meristem formation".Please refer to page 16.

2. The Framework-IV period has taken off after its adoption on April 26 and the launch of its programmes on December 15, 1994. Although the full information packages on the different Activities and Programmes are available from the Commission, an excerpt of information on the programme 'Biotechnologies' can be found in this issue. To stimulate the involvement of PIP members in this area, over 250 European research institutes have received an invitation to team up with PIP members when applying for funds. Details will be published in the Newsletter, or distributed directly.

3. Workshop. The Plant Industrial Platform has taken the initiative to organise a workshop 'defining strategies for information exchange and methods to expedite the application of results', May 10 - 11, Enkhuizen, the Netherlands. There is an increased interest and desire from industry to participate in the development of EU research programmes. What are the possibilities now, are these sufficient, how could a stronger commitment be channelled, what are the timepoints to interact, to what effect would this involvement lead? Results from EU funded programmes generally become available at the time of publication. How could a privileged position for European industry be guaranteed, what would be the benefit for scientists, do we need a structural change? All PIP members receive an invitation and further details on this workshop.

Contents:

Europe: Framework-4 programme
Research Programmes: Project of Technological Priority
The Arabidopsis Genome Project
Molecular Screening News
Research Contributions The Vienna Biocentre
Plant Resistance Genes
Hmc-Network: Gene silencing in transgenic plants
Activities at CPRO-DLO
Member Profiles: R.A.G.T. Seeds Division
S&G Seeds
Activities: Meetings
The PIP Steering Committee

EUROPE


Framework IV Programme (1994 - 1998)


  • In previous issues of the PIP Newsletter general descriptions of the FW-4 Programme and PIP's comments have been presented. The first call for proposals (out of a total of four) is open since January 16, with a deadline for applications on March 24th, 1995. Details can be found in the information packages published by the Commission Services which contain practical information and description of the programmes. Parts of the text below have been taken from the Biotechnology information package and its annex, the workprogramme document.
    The Framework Programme IV contains four main Activities (see PIP Newsletter #4), of which Activity #1 (including RTD and Demonstration Programmes) covers 87% of all expenditures. In this Activity, the area of Biotechnology will be funded with 552 million ECU, which is 35% of total funding in Life sciences and technologies. (which in turn constitutes 15% of total funding in the Activity 'RTD and Demonstration Programmes'). The Biotechnology programme in FW-4 has been selective in choosing three approaches, each one with a specific goal and restricted to identified scientific areas (funding %):

    Concentrated means (375 mECU):
    are employed to harvest highest potential return on task oriented R&D:
    @TABEL2 = Area 1 -
    @TABEL2.1 = Cell factories (22%)
    @TABEL2 = Area 2 -
    @TABEL2.1 = Genome Analysis (16%)
    @TABEL2 = Area 3 -
    @TABEL2.1 = Plant and Animal Biotechnology (24%)
    @TABEL2 = Area 4 -
    @TABEL2.1 = Cell Communic. in Neurosciences (6%)

    Concertation efforts (177 mECU):
    The primary interest is in building upon national research programmes and will form a mix of RTD and Concertation:
    @TABEL2 = Area 5 -
    @TABEL2.1 = Immunology and Transdisease Vaccinology (7%)
    @TABEL2 = Area 6 -
    @TABEL2.1 = Structural Biology (10%)
    @TABEL2 = Area 7 -
    @TABEL2.1 = Pre-normative Research, Biodiversity, Social Acceptance (10%)
    @TABEL2 = Area 8 -
    @TABEL2.1 = Infrastructures. (5%)

    Horizontal activities:
    (integrated in Areas 1-8) are employed to link academic or research institutions and industry (particularly SMEs) in all areas above, essential to the exploitation of the life sciences:
  • Demonstration activities.
  • Biotechnology and society.
  • Socio-economic impacts.
    Most RTD programmes will be carried out through:
  • shared cost actions (typical 50/50 for research programmes).
  • concerted actions (100% funding of costs relating to coordination, travel etc.).
  • preparatory, accompanying and support measures (100% funding).
    Application for project preparatory awards (outline proposals for demonstration projects and for SMEs participating in RTD activities) can be submitted at the first call for proposals of the programme and may cover up to 75% of the costs of the exploratory phase, not exceeding 45 KECU.

    Detailed description of objectives of
    relevant Areas.

    Area 2 - Genome Analysis (Arabidopsis).

    Activities will be divided along three lines of research: sequencing, function search and computer based comparative analysis:

    - The sequencing of regions of the Arabidopsis, B. subtilis and Yeast genomes and of other small genomes of biotechnological interest ( 500Mb) will be carried out by European Networks. In conjunction with other international efforts it is expected that 20% of the Arabidopsis genome and the full description of the Yeast and B. subtilis genomes will be completed by the end of the FW-4 programme. The current Arabidopsis Network (ESSA) is welcome to include appropriate cDNA sequencing activities.

    - The function of unknown genes, sequenced by the European Networks, will be analysed with the help of consortia specialised in the development and distribution of resources, the identification of gene families or in heterologous expression studies.

    - Comparative analysis by computer based interspecies comparison of DNA sequence information will constitute a key to understanding molecular evolution patterns and will ensure the most productive cross fertilisation among the various model genomes.

    Area 3. - Plant molecular and cellular biology.

    The programme is expected to produce a specific advantage by the combination of complementary disciplines and techniques, and to eventually bridge basic science and applications. Examples of combined approaches:
  • Models (Arabidopsis) produce data and technology for use in the genetics of other agricultural species important for Europe.
  • Product-linked research (cellular transport and partitioning, carbon and nitrogen metabolism, sink-source, storage) and technology-driven approaches favoured in industry.
  • Advances in gene cloning, genetic screening, reproductive biology, transgene expression and disease resistance strategies can be put to the service of plant breeding or genetic diversity surveys.
    Proposals originally incorporating a large-scale coordination of numerous participants might be given a pivotal role in implementing a fully integrated action.

    The following research tasks will be implemented:

  • Molecular genetic maps (location of genes of agricultural importance, quantitative trait loci, pest and disease resistance genes, unravelling of function of major genes)
  • Development and morphogenesis (genetic and molecular basis of seedling and flower development, signals in developmentary processes, morphogenesis, cell wall metabolism)
  • Resistance to stress and pathogens (nature of transduction pathways leading to stress tolerance and disease resistance, characterisation of plant genes controlling resistance and pathogen genes coding for pathogenesis factors, synthesis and transmission of plant viruses)
  • Metabolisms (source-sink phenomena, metabolic pathways of plant storage products, regulatory and metabolic pathways during Rhizobium/host plant symbiosis.
  • Gene expression (tissue specificity, stability, sense, antisense, ribozyme technology, homologous recombination).
    Area 7 - Prenormative research: Biosafety and Biodiversity

    In order to ensure the safe applications of Biotechnology, particularly in relation to the commercialisation of new varieties of crops, thorough knowledge is needed of their behaviour in, and interactions with different components of the ecosystem. Examples should be drawn from biological systems of which the understanding is important for future applications. Research will focus on applications which involve unresolved issues related to transgenic plants (pest resistant crops, plants for novel industrial uses), genetically modified microorganisms and transgenic animals. This part of the Biotechnology Programme will not cover research on agricultural and agronomic practices or biological control, nor on nutritional value, consumption or treatment of crops by biopesticides or biofertilisers since these subjects can be found in the Agriculture and Fisheries Specific Programme.
    The following research tasks will be implemented:

  • Microbial Ecology
  • Biofertilizers (symbiotic nitrogen fixing organisms, mycorrhiza-forming fungi, phosphate solubilising fungi).
  • Biopesticides (studies on the genetic basis of interactions, host specificity, persistence)
  • Vaccines
  • Plants (Biosafety aspects, weediness, diversity)
  • Fish
     The development of cellular and molecular methods to detect genetic diversity rapidly and effectively, and to search for variation in specific traits of high economic value, will qualitatively improve European agricultural research and breeding and materially assist conservation of Biodiversity. The following research tasks will be implemented:
    -Development and validation of rapid techniques (RFLPs, RAPDs, VNTR, PCR/sequencing, D/TGGE), sampling strategies, standardisation of data.

    Area 8 - Infrastructures

    Services offered by the activities in this area are meant to have a beneficial impact on all other research areas as described in this programme (especially the Areas 2,6 and 7). Special attention shall be paid to ensure that these services match the research needs, including those of industry and particularly SMEs and will be accessible by proper publicity, wide spread distribution of information contained in databases and other information sources and by providing user-friendly interfaces.

    The following research tasks will be implemented:

  • Information resources on molecular sequences and structures.
  • Taxonomic information and storage databases.
  • A European network of bioinformatics nodes.
  • Horizontal information resources: literature, directories databases.
  • Genetic archives and stock centres.
    Horizontal Activities.

    Demonstration activities
    Can be linked to any scientific and technological research area included in the workprogramme. DA's should prove the technical viability of the new technology, together with, as appropriate, its possible economic advantages. Partnerships executing demonstration projects should have a clear vision of the exploitation of project-deliverables and/or a clear commitment to provide reliable information to public entities such as consumer associations, industrial and professional groupings etc. Extensive information on Demonstration activities was published in the PIP Newsletter #6.

    Ethical, social and legal aspects (Biotechnology and society)
    Commission Services and ad-hoc-working groups will call for transdisciplinary studies (genome research, Biodiversity, intellectual property, transgenics, information technology) where a need is identified for having published expert opinions/reports.

    Public perception (Biotechnology and society)
    Special attention will be given to the development of initiatives to analyse public perceptions in European countries in a worldwide context, through conferences, information papers, reinforcing a debate involving industry, consumers and public authorities.

    Socio-economic impacts.
    Calls for scientific studies possibly highlighting opportunities for Industry and in particular SMEs to benefit from biotechnology research in the development of novel, clean and sustainable products and processes.

    In the evaluation procedure of proposals the Commission will give particular attention to:

  • Transnational collaboration.
  • Scientific and technical excellence, benefits and novelty.
  • Precompetitive character, further development is required to yield marketable products.
  • Potential exploitation of results in a European dimension.
  • The need to encourage the participation of small and medium-sized enterprises (SMEs)
  • The effective commitment of industry to the projects.

    Research

    Programmes



    Project of Technological Priority


    In the PIP Newsletter #4 all PTP research projects (117), divided over 15 themes in 5 networks, have been listed. Furthermore, network C was highlighted in the PIP Newsletter #5. Although some projects have only been active for one year, early results have been obtained. In order to fully serve our readership, and provide opportunity to interact with the theme coordinators, research objectives, a few achievements and address details are presented below. Interim results have been published in a report to the Commission including a 'PTP Coordination Overview' and a 'Scientific Report' over the period November 1993 - July 1994 which is available from the PTP project manager dr. A. Beadle, JIC, Norwich Research Park, Colney, Norwich NR4 7UH, UK, phone:(+44)-603-452571, fax:(+44)-603-4568444

    Embryo pattern formation, development of leaves and seeds, transition of vegetative to reproductive growth.

    Theme A1 coordinators:
     Dr. S.C. de Vries, Wageningen Agricultural University, Netherlands, fax:(+31)-8370-83584.
     Prof.dr. J. Schell, Max Planck Institut, K”ln, Germany, fax:(+49)-221-5062-213.
    Objectives and results: There are roughly 3 major areas of research in this theme:
  • a study of the formation of the epidermis in embryo development, making use of somatic as well as zygotic embryos and an analysis of seed development.
  • a study of leaf formation including aspects of light control.
  • a molecular-genetic analysis of genes involved in various aspects of flower development.
     Research involves the use of Arabidopsis, Antirrhinum, Daucus, Linum, Pteridium, Hordeum and Solanum as plant species.
  • In situ hybridisation, employing the epidermis-specific marker gene AtEP2 and Arabidopsis embryo mutants disturbed in epidermis formation, showed that in mutant embryos the expression pattern deviates from normal, confirming the morphological observations.
  • Screening of T-DNA and En/1 tagged Arabidopsis lines has resulted in the identification of several lines that exhibit reduced seed dormancy.
  • The possibility to use transgenic plants overexpressing phytochrome A genes as a means to eliminate the shade avoidance reaction and as a result of that to increase the amount of harvestable compounds is studied by several groups.
    Molecular mechanism of action of plant growth factors: from primary perception to gene expression.

    Theme A2 coordinators:
    Prof.dr. M.A. Hall, University College of Wales, Aberystwyth, UK, fax:(+44)-970-622-350.
     Prof.dr. J. Schell, Max Planck Institut, K”ln, Germany, fax:(+49)-221-5062-213.
    Objectives and results: Plant growth factors and a variety of environmental stimuli are the major signals that influence plant growth and development. Whereas a number of small organic molecules have been identified as "plant growth hormones" and have been shown to affect a great diversity of the developmental responses in plants, their precise mechanisms of action are still largely unknown. The main purpose of the research to be coordinated in this theme is the identification of the various elements that play a role in mediating the activity of plant growth factors from their primary perception by still largely hypothetical receptors all the way to differential gene expression.
  • A major objective focuses on auxins and the studies here will embrace work both on genes and their products involved in auxin biosynthesis and in its release from intracellular conjugate pools (especially the rol genes) as well as studies on the auxin perception and transduction mechanisms. Within the area of perception, studies will concentrate on the identification, localisation and cloning of putative membrane-bound and soluble receptors. The question whether GTP-binding proteins play a role in auxin signal transduction will be investigated.
  • In parallel studies using molecular genetic approaches, 'gain of function' gene tagging will be used to obtain mutants affected in the mechanism of action of auxin and other plant growth factors.
  • In related work the role of jasmonic acid and abscisic acid in wounding will be investigated via approaches involving investigations on the biosynthesis of the former, the use of 'antisense' technology and studies on 'wound-induced' genes.
    Biochemical and physiological mechanisms of action of xyloglucan oligosaccharides: novel plant growth factors.


    Theme A3 coordinators:
     Dr. S.C. Fry, University of Edinburgh, UK,
     fax:(+44)-316-505-392.
     Prof.dr. R.B. Flavell, John Innes Centre, Norwich, UK, fax:(+44)-1603-456-844.
    Objectives and results: Xyloglucan, a major hemicellulose of the primary cell walls of higher plants, can be cleaved enzymically to yield specific fragments (xyloglucan oligosaccharides; XGOs) some of which possess growth-regulating activities. The hypothesis has been proposed that those XGOs that promote plant cell expansion do so by acting as substrates for the newly discovered enzyme, xyloglucan endotransglycosylase (XET). XET is believed to participate in the mechanics of cell expansion and also in the cell-cell separation that occurs during fruit ripening. XGOs will also be tested in their other known biological effect, viz, the inhibition of gibberellin-, auxin- and H+ -stimulated growth.
  • A reproducible assay for growth promoting and inhibiting effects of XGOs has been devised. It has been shown that growth-regulating XGOs are not elicitors of defence-related responses.
  • Work has begun on the preparation of a library of both 3H-labelled and non-radioactive XGOs, for use in both biological and enzymic studies. These XGOs are being produced from the xyloglucans of diverse plants, including both Dicots and Monocots, and appear to include some novel structures.
  • Numerous Pisum growth bioassays have been conducted and although these sometimes show predicted growth-regulating effects of XGOs, this is not always the case. Improved reproducibility of the bioassays is identified as a goal.
  • A novel Pisum bioassay has been developed in which whole excised epicotyls are incubated with their bases in solutions of XGOs, such that transpiration can facilitate the delivery of the XGOs to the major growing zone of the stem.
    Hormone-mediated regulation of cellular development in the moss, Physcomitrella patens (EUROMOSS).

    Theme A4 coordinators:
    Dr. T. Wang, John Innes Centre, Norwich, UK,
     fax:(+44)-1603-56844.
     Professor R.B. Flavell, John Innes Centre, Norwich UK, fax:(+44)-1603-456-844.
    Objectives and results: 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.
  • Reproducible protocols for LM, SEM, TEM have been established for the investigation of protonemal growth in wild-type and mutant strains.
  • Tritiated adenine and isopentenyladenosine have been fed to wild type and mutant tissues to establish their patterns of cytokinin metabolism.
  • Several cDNA libraries have been constructed. These are from different stages of the wild type and from tissues treated with cytokinin for different times.
  • Both PEG and microprojectile techniques have been established for moss. Several plasmid DNA copies are incorporated at a single locus in stable transgenes. Several lines of evidence indicate that unstables are extrachromosomal replicative transformants existing as high copy number concatenates.
    Perception and signal transduction of lipo-oligosaccharides of Rhizobium.

    Theme A5 coordinators:
     Dr. T. Bisseling, Wageningen Agricultural University, Netherlands, fax:(+31)-8370-83584.
     Professor M. van Montagu, University of Gent, Belgium, fax:(+32)-92-645-349.
    Objectives and results: The Rhizobium-legume interaction offers a way to elucidate a signal perception-transduction pathway in plants because of the availability of specific triggering molecules (the natural Nod factors and modified ones) and responding plant genes with relatively early expression such as enod40. New data on structure determination of Nod factors produced by a variety of Rhizobium strains confirm the general picture established previously that the molecules are chitotetra-or pentamers, acylated at the non-reducing end and carrying different classes of substitutions.
    Major questions are:
  • how (where) is the Nod factor signal perceived by the plant host?
  • what happens subsequently at the level of plant gene expression and plant protein activity?
  • are similar molecules produced by plants themselves to play a role in development?
    Isolation and characterisation of genes relevant to abiotic stress.

    Theme B1 coordinators:
     Dr. D. Bartels, Max Planck Institut, K”ln, Germany,
     fax:(+49)-221-5062-213.
     Prof.dr. F. Salamini, Max Planck Institut, K”ln, Germany, fax:(+49)-221-5062-413.
    Objectives and results: The purpose of the research combined in this team is to understand how plants respond to environmental factors and to develop and test strategies for improving plant productivities under adverse conditions. Isolation of structural genes and identification of regulatory elements positively contributing to stress resistance and stress tolerance is the main objective. Sources for the isolation of structural genes and regulatory elements are model plants (Arabidopsis, Craterostigma) and crop plants (maize, barley, legumes).
  • Mutants affecting the stress phenotype or the signal transduction pathways were isolated from Arabidopsis. Currently more than 20 early lines and more than 40 late lines have been isolated in different screenings. These lines are being organised in phenotypic groups that are subjected to complementation analysis, analysis of dominance and backcrossing. Furthermore, crosses to map the corresponding mutations are performed and the acclimation response is characterised.
  • Stress responsive genes have been isolated from different stress situations and from different plant species. The cDNA designed AF93 represents a gene inducible under cold condition and drought stress. The cDNA clone T59 is homologous to mRNA expressed only under low temperature conditions in leaves. By utilising yeast as experimental system three genes (HAL1,2 and 3) have been isolated which by overexpression improve salt tolerance.
  • To unravel the signal transduction cascades in stress reactions several approaches are used. In transient expression assays it was found that the superoxidedismutase-GUS and the Apx-GUS fusions were induced by various, structurally unrelated thiomolecules, suggesting a redox-control. Besides the direct promoter analysis to identify signalling molecules a reverse genetic approach is being used for the heat shock response.
    Molecular and cellular studies of the role of membrane transport processes in plant responses to environment and to abiotic stresses, including salinity and potassium deficiency.

    Theme B2: coordinators:
     Prof.dr. R.A. Leigh, Rothamsted, Harpenden, UK, fax:(+44)-582-760-981.
     Prof.dr. F. Salamini, Max Planck Institut, K”ln, Germany, fax:(+49)-221-5062-413.
     Objectives and results: The aim is to identify and characterise Na+ and K+ transporters in order to determine their role in cellular responses to K+-deficiency and salinity, and to understand the physiological role of individual members of the plasma membrane ATPase multigene family. In studies of K+ and Na+ transport, the intention is to identify and characterise transporters and their genes using electrophysiology, by complementation of yeast transport mutants and by heterologous expression in yeast or Xenopus oocytes. At the cellular level, the aim is to understand the role of the yeast HAL1 gene in salinity tolerance and the processes that contribute to the homeostasis of cellular K+ and Na+ concentrations plants. Other work on the plasma membrane ATPase multigene family is aimed at understanding the role of individual family members including their tissue-specific expression, their functions in coleoptile elongation and in root nodules of legumes, and the determination of their biochemical properties including the role of the auto-inhibitory C-terminal domain.

    A strategy to characterise the QTLs involved in drought tolerance in maize.

    Theme B3 coordinators:
     Prof.dr. D. de Vienne, INRA-CNRS-UPS, Gif sur Yvette, France, fax:(+33)-1-6941-2790.
     Prof.dr. F. Salamini, Max Planck Institut, K”ln, Germany, fax:(+49)-221-5062-413.
    Objectives and results: RFLP maps constructed from segregating populations allow loci controlling quantitative traits (QTLs) to be detected, and their effects to be estimated. This approach is applied to agrophysiological traits responsive to water deprivation, using not only anonymous probes, but also cDNAs of genes induced by water-stress as RFLP probes. The expectation is that the polymorphism of these genes may result in quantitative variation of drought response traits. All the traits responsive to drought stress, from the molecular to the agronomical level, display genetic variability.
  • A mild water deficit induces more changes in carbohydrate metabolism than in leaf photosynthesis, transpiration and growth. -Novel drought responsive proteins have been found.
  • A protein previously described in tomato and known to be ABA and stress-responsive has been characterised.
     In addition to insights into the genetic basis of the drought responses, the project should provide 'candidate' genes for further molecular analysis, marker-assisted selection and, in the long run, transformation.

    Development and differentiation of starch-storing organs. The potato tuber as a model system.

    Theme C1 coordinators:
     Dr. D. Vreugdenhil, Wageningen, Netherlands, fax:(+31)-8370-847-40., Prof.dr. D. von Wettstein, Carlsberg Laboratory, Copenhagen, Denmark, fax:(+45)-3327-4766.
     Objectives and results: It is the objective of the collaborative effort to clone and characterise the genes which are turned on when stolons differentiate into tubers. It is a further aim to clone the structural genes for all proteins/enzymes required for starch granule synthesis, so it can be duplicated in the test tube. Molecules that regulate partitioning of starch and sucrose synthesis are to be overexpressed or suppressed using transgenic plants with appropriate sense and antisense gene constructs.
  • The model system for tuberisation in potatoes, viz in vitro tuber formation on single-node cuttings, has been optimised, tuber formation occurs highly synchronised and predictable.
  • Anatomical and morphological studies on the various stages of tuber formation have been done. A good correlation was observed between the orientation of the cortical microtubules in cells in the axillary bud and the change in the direction of cell division and expansion at the onset of tuber formation.
  • Protocols for the preparation of a PCR subtractive library from tuberising stolons have been optimised to produce a library. Also daily stages of tuberising material were subjected to poly-A+RNA isolation, from which cDNA was synthesised. Currently transcript are being identified which show modulated gene expression during the tuberisation process.
  • Methods were developed to determine the activities of most enzymes involved in the conversion of imported sucrose into starch, the measurement of the main metabolites and also proteins were isolated from starch granules for the production of antisera to screen potato tuber-specific expression libraries.
    Redirecting carbohydrate flow in plant cells.

    Theme C2 coordinators:
     Dr. J.C. Smeekens, University of Utrecht, Netherlands, fax:(+31)-30-513-655.
     Prof.dr. D. von Wettstein, Carlsberg Laboratory, Denmark, fax:(+45)-3327-4766.
    Objectives and results: A molecular approach is taken to study and modify carbohydrate interconversion processes in plant cells with the aim to produce modified or new carbohydrates and other metabolites in plants. Specifically the theme is focusing on aspects of the following processes:
  • translocation of metabolites across biological membranes.
  • diversion of carbohydrate flow into amino acid and cell wall biosynthesis.
  • metabolism of sucrose and fructans.
  • development of technology for gene expression in crop species and analytical methods for carbohydrate analysis.
     In the first year the translocators have been cloned and the functional analysis is well underway. 2-OG metabolising enzymes are being purified and analysed with emphasis on isocitratedehydrogenases. Regulatory steps in carbohydrate interconversion have been identified by (over)expressing a range of enzymatic activities in plants which affect carbohydrate partitioning or by suppressing endogenous enzymatic activities.
    Objectives for the next 36 months are:
  • the functional characterisation of triose-phosphate translocators from C3 and C4 plants and the analysis of their role in partitioning processes.
  • The role of 2-oxoglutarate in amino acid assimilatory processes.
  • Understanding the regulatory steps in intermediary carbohydrate metabolism, especially with respect to diversion of carbon to cell wall biosynthetic processes.
  • The role of invertases in sucrose metabolism. The redirection of sucrose in fructan polymers and metabolism of fructans in plants.
  • Methodology for structural determination of complex fructans and the identification of sink specific promoters for expression of genes involved in carbohydrate metabolism in crop plants.
    Genes and enzymes for carotenoid biosynthesis: structure, regulation and heterologous expression.

    Theme C3 coordinators:
     Dr. P. Bramley, University of London, UK, fax:(+44)-784-434-326.
     Prof.dr. D. von Wettstein, Carlsberg Laboratory, Copenhagen, Denmark,
     fax:(+45)-3327-4766.
    Objectives and results:
  • Cloning carotenoid genes from cyanbacteria, fungi and higher plants and the D6-desaturase form Borago.
  • Elucidation of rate-limiting steps in carotenogenesis in higher plants.
  • Characterisation of the carotenogenic enzyme complex in plastids and elucidation of the reaction mechanisms.
  • Purification of carotenogenic enzymes, antibody production and screening of expression libraries.
  • Heterologous expression of carotenoid genes in tomato, tobacco, pepper and expression of D6-desaturase to tobacco and rapeseed.
  • Production of crop varieties over-producing carotenoid or g-linolenic acid.
    Metabolic regulation in the developing seed.

    Theme C4 coordinators:
     Dr. R.D. Thompson, Max Planck Institut, K”ln, Germany, fax:(+49)-221-5062-413.
     Prof.dr. M. Koornneef, Wageningen Agricultural University, Netherlands, fax:(+31)-8370-83146.
    Objectives and results: The participants in this programme are working on various aspects of the regulation of storage product accumulation in cereal seeds and objectives include the isolation of new genes and characterisation of the roles of known genes such as Opaque-2 and RKin-1.
  • The promotor of the C-hordein gene of barley has been shown to increase in activity in response to elevated levels of reduced nitrogen compounds. This nitrogen response is primarily mediated via a cooperation of the GCN4 and endosperm promotor motif. Opaque-2 was tested for its possible involvement in a nitrogen response of the 22kD zein promotor.
  • The yeast SNF1/SNF4 gene complex represses at the transcriptional level enzymes of carbohydrate synthesis, raising the possibility that the cereal grain SNF1 homologue may also associate with a second regulatory component. As well as a role in carbon catabolite repression in plants, there are indications that the pathway may be involved in certain stress responses.
  • The isolation of new genes involved in zein regulation by transposon tagging and by complementation of yeast mutants is undertaken, as well as the isolation of a series of mutants defective in protein secretion.
    Molecular strategies to modify nitrogen carbohydrate partitioning in crop plants.

    Theme D1 coordinator:
     Prof.dr. M. Caboche, INRA-Versailles, France,
     fax:(+33)-1-3083-3099.
    Objectives and results: Genes contributing to the transport and utilisation of N or C metabolites will be identified. Transgenic plants in which the expression of these genes will be modified (overexpression, antisense inhibition, tissue specificity of expression modified) will be obtained and characterised by molecular, biochemical, physiological and ecophysiological approaches.
  • The PCR-based identification of Nicotiana genes to Chlamydomonas nitrate transporters has so far not been successful. An alternative approach based on the differential screening of nitrate inducible transcripts is being developed.
  • An inventory of nitrate transporters is made in Chlamydomonas. Mutants lacking either of Nar1, Nar2, Nar3 or Nar4 genes have been obtained. Their analysis shows that Nar2 is essential for the transport of nitrate, but not nitrite.
  • Transgenic tobacco and potato plants inhibited for the plastidiary fructose 1,6 bisphosphatase have been obtained via an antisense-mediated inhibition. These transformants are under characterisation. In parallel, transgenic potato plants have been created which due to antisense mediated inhibition haven heavily reduced levels of two major tuber storage proteins.
  • Transgenic tobacco plants in which nitrate or nitrite reductase expression has been inhibited by co-suppression are under characterisation. In these plants a reduction of chlorophyll biosynthesis is detectable when they are supplied with nitrate, as opposed to ammonium.
  • Differential assays to separate phosphorylated and non-phosphorylated SPS and NR have been adapted to tobacco. Nitrate itself regulates root-shoot allocation independently of its metabolism. Nitrate is also an extremely powerful up-regulator of anaplerotic metabolism. Feedback mechanisms adjusting nitrate assimilation to GIN/Glu ratios comes into operation to small changes of NR activity.
  • Three different families of proteins that are capable of mediating uptake of amino acids in yeast transport mutants have been identified. Their expression patterns are under study, using gene reporter techniques. Experiments are in progress to identify such amino acid transporters expressed in potato tubers.
  • Potato transformants expressing constitutively a nitrate reductase gene from tobacco have been produced. In parallel, a nitrate reductase gene from potato has been cloned and will be used once it has been further characterised to develop an antisense strategy against the expression of leaf potato nitrate reductase.
  • Characterisation of plants constitutively expressing nitrate reductase has shown that although increased glutamine contents were observed in these plants, this increase has no consequences as regards total protein and biomass production, which remained unchanged. In contrast the constitutive expression of SPS resulted in large changes in carbon partitioning and biomass production.
    Investigating the regulation of the nitrate assimilatory pathway in a higher plant through genetic manipulation and mutagenesis.

    Theme D2 coordinators:
     Dr. B. Forde, Rothamsted Exp. Station, Harpenden, UK, fax:(+44)-582-76081.
     Prof.dr. M. Caboche, INRA-Versailles, France,
     fax:(+33)-1-3083-3099.
    Objectives and results: To improve the nitrogen-use efficiency of crop plants, the regulation of the pathway is investigated by which nitrate is taken up from the soil and assimilated by the plant. Novel genes that may be important in determining nitrogen-use efficiency will be isolated.
  • Clones for the major/sole cytosolic glutamine synthetase from nodule and root, and the single nitrate reductase have been isolated from L. japonicus cDNA libraries. Antisense constructs have been made and transformants will be tested.
  • Screening for chlorate-resistant mutants, affected in leaf nitrate accumulation and photorespiratory mutants is undertaken.
  • Eight hundred transgenic lines of L. japonicus carrying Ac or Ds have been generated

    The Arabidopsis Genome Project


    Progress and Exploitation

  • Coordinator: Dr. Michael Bevan, John Innes Centre, Norwich, UK.
    The EC-funded Arabidopsis Genome Project began a pilot-scale operation aimed at sequencing 2 million base pairs (Mbp) of the genome in late 1993, and it is intended to complete this initial phase of the project by late 1996. In the next three years (1996-1999) the EC have indicated the possibility of extending this pilot-scale project into production-scale sequencing of more than 10 Mb, focusing on completing a major portion of a chromosome. This EU effort will be accompanied by a similar contribution from US colleagues over that period, such that by the end of 1999 over 25% of the estimated 100 Mbp genome would have been completed. The remainder of the genome will then be finished by 2004, probably using methods which are still being developed as part of the Human Genome Project. The complete sequence of a genome will have a variety of applications. Presently the most worthwhile application comes from the extensive and comprehensive physical maps, comprising YAC (yeast artificial chromosome) contigs with multiple redundancy, which now populate the 5 chromosomes of Arabidopsis. It is estimated that a near complete coverage of single-copy sequence will be available in 12-18 months.
    With this information, scientists aiming to use map-based methods for gene isolation will be able to accomplish this task, which previously took years of tedious and uncertain chromosome walking, by reference to a stock centre database to obtain mapping populations and YAC contigs. If the desired trait maps within a sequenced area then fine structure mapping using probes from the sequenced regions and complementation with sequenced cosmids should identify the desired gene readily.
    In the short to medium term this acceleration of the genome walking process should, if the appropriate bioinformatics tools and stock centres are used, lead to a more extensive use of Arabidopsis as an experimental system, and stimulate a wider variety of biological questions to be asked in plants.

    Allied to these developments in Arabidopsis, comparative genome analysis between well characterised model species and related crop plants will permit identification of crop plant homologues of well described Arabidopsis genes. A compelling example of the power of developing systematic links between Arabidopsis and Brassica is provided by the recent observation that a gene isolated in Arabidopsis called CO, a transcription factor which regulates flowering time in relation to daylength, maps to three collinear regions of the Brassica nigra genome, two of which form QTLs (quantitative trait locus) for flowering time. In another example, it has emerged that the complex profiles of glucosinolates, which are major secondary metabolites in Arabidopsis and Brassica, are probably determined by homologous gene systems. Arabidopsis RFLP markers flanking the Gsl-elong gene (which regulates elongation of the aliphatic side chain) also map near to a Brassica locus controlling the same trait. Glucosinolates determine the flavour of Brassica vegetable and salad crops, the quality of oilseed rape meal and also mediate pest and pathogen interactions, making these genes important targets for manipulation. The provision of integrated mapping information can still be used to facilitate positional cloning of agronomic genes in Brassica even in the absence of characterised homologues in Arabidopsis. In such cases, the principle of collinearity and the availability of ordered physical libraries can be combined to supply a high density of probes for the fine-mapping and isolation of the Brassica gene. Similar approaches are being considered for cereal genomes, based on the YAC contigs of the rice genome being assembled. It may be possible to determine possible collinearity between rice and Arabidopsis in the future, thus spanning distantly related plants and providing a framework for establishing syntenic relationships between many dfferent plant species.

    The depth of knowledge being obtained in Arabidopsis concerning fundamental aspects of growth, development and pathogen resistance, coupled to the extensive partial cDNA sequences available for rice and Arabidopsis, is already being exploited by identifying possible rice homologues of Arabidopsis genes. A major expectation of systematic genome sequencing, based on the data obtained from both the yeast and nematode genome sequencing programme, is that novel classes of genes will be identified which may never have been able to be identified in standard mutagenesis screens, possibly because the resultant phenotypes may be lethal or that potential redundancy or compensatory mechanisms are activated upon the loss of function of certain genes. At least 30% of the potential genes identified in yeast, perhaps one of the most intensively studied organisms, have an unknown actual or predicted function. With the imminent completion of the 14 Mb sequence of the yeast genome, the major emphasis of the yeast genome project will now turn to a systematic attempt to ascribe a function to most potential yeast genes uncovered by sequencing. Similar function search activities are planned to accompany genomic sequencing work in Arabidopsis in the near future.

    The function search network(s) associated with Arabidopsis will focus initially on methods development, aimed at establishing materials needed to apply transposon- tagging and T-DNA tagging methods developed in the BRIDGE programme and elsewhere to sequenced regions of the genome. This programme will have many interesting candidate genes from which to choose once the plant lines have been assembled, and it is at this stage of the genome programme that results most immediately applicable to the plant biotechnology and agricultural industries will be obtained. But these developments are not yet a part of an EC-funded scientific network, and if they are funded, they will not begin to systematically produce mutations until 1996.

    We need to consider how the sequencing programme can benefit relevant EU industry now, and to develop structures that promote the efficient use of sequence data. Several things need to be done. The first is to explain ownership and public distribution of sequence data to industry, the second is to demonstrate the type of information which is produced by a sequence programme, and the third is to set this information in a context of interested scientists and industry together with the appropriate means of exploiting the interesting gene(s). Firstly, participants in the EC-funded sequencing project who produce sequence are the owners of that sequence until it is made publically available. Other participants can only gain access to that sequence via the MIPS (Martinsried Institute for Protein Sequence) Informatics Node with the owner/originator's permission. Sequence is first submitted to MIPS, where it undergoes checks for conformity to the restriction map, accuracy tests in overlap regions and is finally analysed. Until the network begins to sequence in full-scale production mode, submission of sequence will not occur regularly. It is only after this analysis has been carried out, and the maximum number of putative open reading frames (ORFs) and other interesting landmarks identified, that the sequence will be published and submitted to public databases. This delay will enable the partners in the sequencing network to use the unprecedented size of their contiguous sequences to apply and develop the most useful analytical tools for their sequence. The sequence will only be of potential value until this analysis has been completed to the best of our ability, and it is at this stage that PIP members will be given a summary of the analysis and a list of the originators of that sequence. It is hoped that this process will begin towards the end of 1995 and continue on a regular basis. Future release to PIP and public release will be entrained to publication of the sequence by members of the sequencing network.

    In addition to collaborative publication of large contiguous regions, individual laboratories in the network will be able to independently publish sequence originating from their labs at any time, but it has to first be verified and accepted for funding by MIPS before publication. The data produced by genomic sequencing is complex and voluminous, and is most readily accessed and displayed using databases such as AAtDB, but a paper version will be made available to PIP members by the Coordinator. The information provided will mostly comprise lists of coordinates of identified ORFs and BLAST (basic local alignment search tool) scores showing the probability of a match between all six frames and known proteins from all other organisms in the latest release of sequence databases such as EMBL and Genbank. The challenge facing informatics experts at the moment is to identify ORFs in genomic sequence. This can be done by matching partial cDNA sequence from Arabidopsis or rice, for which there are a considerable number (but still only 20-30% of the expected total), or less successfully, cDNA sequence from non-plant sources. There are tools such as GRAIL, GENEFINDER and GENEID, which are neural networks capable of being "trained" to recognise coding regions when fed with sufficient cDNA and other examples of coding sequence. A preliminary analysis of two sequenced cosmids in the FCA region of chromosome 4 using GRAIL and BLAST shows a potential gene density of one every 4-5 kb, indicating that the total number of genes in Arabidopsis may be in the range of 20-25,000. It is interesting to note that ORFs recognised by matches to ESTs (expressed sequence tag) have not been recognised by GRAIL. To overcome this the Informatics Node of the EU Arabidopsis Sequencing Network is training GRAIL to recognise Arabidopsis ORFs using the 7000 publically available Arabidopsis EST sequences. An example of a similarity search of two cosmids sequenced at JIC using BLAST is shown in the Table (see insert on the previos page). Based on these preliminary data, and asuming a relatively even distribution of gene density (which is not the case based on preliminary data from the network) we predict that by the end of the pilot-scale effort in mid-1996 400 ORFs of unknown function, and about 350 ORFs corresponding to genes of known function in other organisms, will have been identified. Continued refinement of the GRAIL analysis system may permit further ORFs to be identified with reasonable assurance.

    The third necessary precondition for PIP members to obtain maximal benefit from the sequencing programme is to develop and maintain communication with a wide variety of scientists working in fields in which EU industry is interested. The sequence data should be viewed as an enabling tool which biologists can use to isolate and characterise genes regulating agronomically important traits, and it is through these biologists that maximal mutual benefit can be obtained. It is noteworthy that many of the projects in plant science and agriculture described in the Workplan of Framework 4 could (or more emphatically should) use the power of Arabidopsis genetics to understand and manipulate a wide variety of processes in plants. Setting up a functional analysis network will provide a key link between the sequencing programme and exploitation by EU biologists and industry. One way of ensuring that appropriate communications are set up between the scientists involved and PIP would be for an umbrella organisation composed of the Coordinators of the relevant EC-funded projects in Framework 4 to interact to ensure maximal use of genome sequence and interact with PIP to ensure maximum benefit can be obtained from the integrated EU-wide network of plant scientists. For further information please contact: Dr. Mike Bevan, fax:(+44)-1603-505725 E-mail: bevan@bbsrc.ac.uk

    Molecular Screening Tools


  • Coordinator : Angela Karp, IACR-Long Ashton, Department of Agricultural Sciences, University of Bristol, Long Ashton, BS18 9AF, U.K.
    Highlights of the generic meeting at Ede, the Netherlands, November 16-19, 1994. Background to the meeting and general conclusions. The major objectives of the meeting were to: (1) assess current progress (2) discuss whether we were on line with the objectives of the programme (3) to reappraise emphasis in the current programme and to consider future emphasis (eg, for Framework IV).
    The reproducibility of RAPDs. RAPD reproducibility in lab networks was put to test (see below). The general feeling was that RAPDs are acceptable for people working in their own labs, in which case you could pursue a diversity study to its conclusion using this approach, but they are not suitable for a network system in which data is sent into a central pot. They therefore have limited use in this programme (see also general conclusions).
    The use of tools in studying diversity in natural populations of plant and animals. In this part of the programme it was felt important to have both of the following two directions of research: (1) Identification of those sequences (nuclear and/or organellar) whose behaviour, in terms of their rate of change, mode of inheritance and evolution, mean that they can be used to study diversity at the below species level, (2) Demonstration and development of strategies for using these sequences to study the genetic structure and function of populations and the way that diversity changes both temporally and geographically. In fact, all this information is needed for conservation. Unless we know enough about the sequences themselves we cannot reliably use them as a tool for studying diversity in living populations and, equally, we need to have as quickly as possible information on the genetic structure, and temporal and geographic variation in populations for conservation decisions.

    The use of the tools in screening accessions and in classification. It was felt that we cannot make a sensible decision about whether or not to conserve something (whether we are talking of natural populations or collections) simply based on differences in the levels of diversity if we do know what the phylogenetic relationships are. Phylogenetic concepts are important for providing a framework of understanding on the significance of the genetic diversity studies. Phylogenetic relationships also reveal how long it has taken for the species to evolve - which is also important for conservation decisions. The general feeling, therefore, was that we need to know both how much genetic variability there is in populations, accessions and collections and also their phylogenetic relationships - since the latter may be equally crucial for conservation decisions.

    The development of universal tools. Of the three approaches being used the group felt that most emphasis should be placed on the development of probes and primers, which can be used in different species, and on methods and protocols that can speed up the identification of the desired sequences. There seems to be general agreement that some types of markers are more appropriate to population studies than other types, and the problem is that the ones that are more appropriate are also more difficult to identify. We know that we won't be able to develop primers for all the single species, so what we should be able to do at the end is to have protocols that will allow other people to do this in a faster and easier way. The comment was made that conserved primers are at risk of picking up contaminant microorganisms for example. Furthermore, in plants, 50% or so are amphiploids and therefore primers may amplify several loci. In our definition of "Molecular Screening Tool" we recognise that tools could also be the quickest method of finding something, eg a fast way of constructing a library enriched with the target sequences, eg microsatellites.

    General conclusions reached so far: It was felt that if we are developing techniques for wide-scale screening for conservation decisions or for evaluation of diversity by agriculture and associated industries, we need techniques that are reproducible, comparable and easily handled by digital means. We don't find that RAPDs fit the bill and we don't even find them easy. With RFLPs we get indirect information. Therefore, the feeling of the group was that we should concentrate on sequence information. But one cannot rely on sequences only, since we don't yet have enough nuclear genes to work from, sequencing 1000s of individuals is still not realistic and also because sequence data does not adequately cover the whole genome. Therefore, we should also use microsatellites. It was generally felt that a single tool (or class of tools, eg. ITS or microsatellites, or an anonymous sequence) would not give an accurate enough measure of diversity. There are some advantages, particularly in population studies, in using an organellar and a nuclear marker since their congruency, or lack of congruency will shed light on behavioural and or dispersal factors. It was therefore the group's feeling that future focus should be on identification of microsatellites and, particularly, on the development of rapid protocols for their identification in nuclear and organellar DNA, and on the development of PCR- sequencing approaches, including fast assays of sequence divergence (eg, PCR-RFLP, TGGE, DGGE, SSCP, heteroduplex formation etc).

    There is still a need for more automation, and for further development of the technologies (such as those listed above) for rapid detection of sequences and differences contained within them. The group would welcome any feedback from PIP members on these comments and we are actively seeking industrial partners for applications in Framework IV.

    For more information please contact dr. A. Karp, phone:(+44)-275-392-181, fax:(+44)-275-394-281

    Research

    Contributions



    The Vienna Biocenter


  • Prof.dr. Erwin Heberle-Bors, Section of Plant Genetics, Vienna Biocenter, Institute of Microbiology and Genetics Dr.Bohrgasse 9,A-1030 Vienna, Austria
    The Vienna Biocenter consists of five university institutes of the Medical and Science Faculties of the Vienna University and the Institute of Molecular Pathology (owned by Boehringer Ingelheim, headed by Max Birnstiel). We have initiated activities devoted to plant research (including cytogenetics, molecular plant pathology, Arabidopsis genetics). We welcome industrial partners to join our efforts.

    Pollen development. A highly efficient pollen system for in vitro maturation of tobacco pollen from microspores (less good in wheat) has been developed, which can be used for the rescue of sterile pollen, rescue of self-incompatible pollen, for pollen selection and for pollen transformation. Pollen selection can speed up the breeding process by increasing the frequency of target genes in the progeny. Pollen transformation via in vitro pollen maturation does not require regeneration and requires only a few days of in vitro culture. We intend to fully explore the biotechnological potential of this novel in vitro culture system.

    Microspore/pollen embryogenesis. A highly efficient pollen culture systems for the regeneration of haploid and doubled haploid plants in tobacco and wheat has been developed. We have shown in tobacco that this system can be used for plant transformation and are conducting similar work in wheat. In our wheat pollen cultures, doubled haploid production is genotype-independent (collaboration with F. L”schenberger, Probstdorfer Saatzucht Ges.m.b.H). For quality control of doubled haploids, we are studying the cytogenetics of aneuploidy (flow cytometry) and the origin of albino formation (mapping plastid genes) in pollen plants of wheat. We are studying the cell biological and molecular mechanisms of embryogenic induction in tobacco pollen (role of cell cycle and signal transduction genes during induction).

    Pollen allergy. In collaboration with medical people in Vienna, we are studying two allergic pollen proteins of birch, Betv1 and Betv2. One protein is homologous to a class of PR-proteins, of which a large family of homologous birch genes has been isolated. Expression of some genes is pollen-specific, others are inducible by pathogen attack. We therefore have clear evidence for a hitherto unknown biochemical function of this class of proteins. The second protein is a profilin, an actin-binding protein, involved in the dynamics of cytoskeletal rearrangements during development.

    Signal transduction. From tobacco and alfalfa, we have isolated a number of mitogen-activated protein kinase genes (which in animals have been shown to signal hormone-receptor binding and stress attack to the nucleus, MAP and SAP kinases). By using antibodies prepared against the recombinant proteins we are studying changes in protein kinase activities is response to various exogenous signals, including hormones, stress factors, pathogen attack, and during pollen development. In addition a calcium-dependent protein kinase is studied.

    Cell cycle genes. The cdc2-protein kinase is the principal cell cycle regulator in eucaryotes whose activity is controlled by the regulatory cyclins, inhibitory proteins and by phosphorylation. We have isolated a number of cdc2 and cyclin genes, also a nucleolin gene, from alfalfa (cdc2-genes also from tobacco) and are studying their expression in synchronised cell suspension cultures and in the plant (in situ hybridisation, Northern, Western, protein kinase assays, promoter-GUS-fusion genes in transgenic plants). We want to know how the plant cell cycle, during development, is regulated by upstream factors such as hormones. A focus is the reactivation of the cell cycle in quiescent cells cultured in vitro (alfalfa somatic embryogenesis, tobacco microspore embryogenesis).

    EU-funded research projects. A proposal for funding is being prepared on doubled haploids from pollen, with emphasis on cereals, Solanaceae and Brassicaceae (vegetables). Goals are genotype-independent doubled haploid production via isolated pollen culture, the role of the initial developmental stage and stress factors for embryogenic induction, quality control (spontaneous diploidization, aneuploidy, albinism), and transformation. Somatic embryogenesis could possibly be included in this proposal. A second proposal would involve pollen transformation and should focus on a specific breeding goal. We are also involved in a proposal on plant cell cycle regulation and our work met interest among colleagues working on proposals for programmes on male and female reproductive biology and embryogenesis.
    We are also interested in partners working on signal transduction and on pathogen defence. For further information, please contact: Prof.dr. Erwin Heberle-Bors, fax:(+43)-1-79515-4114, E-mail: erwin@gem.univic.ac.at

    Plant Resistance Genes


  • Dr. Charlotte Grimm, Institute of Botany, Dept. of Cytology and Genetics, Rennweg 14, A-1030 Vienna, Austria.
    In an attempt to search for specific plant resistance genes against phytopathogenic bacteria we have recently isolated a new gene from Arabidopsis thaliana that is highly regulated (W. Aufsatz and C. Grimm, Plant Mol. Biol. 25, 229-239, 1994):

  • The gene (CXc750) is expressed in an ecotype specific manner. As far as analysed yet, the expressing ecotypes are resistant against certain Xanthomonas campestris pv. campestris (Xcc) strains.
  • Although constitutively expressed, the gene is also regulated in response to infection with these avirulent Xcc strains, i.e. we observe an increase of transcription by a factor of 7 within 24 hours post-infection. In contrast, this response is not observed after infection with avirulent Pseudomonas strains or virulent Xcc strains.
     Simple wounding leads to dramatic down regulation of gene expression.
  • The non-expressing ecotypes of A. thaliana contain a silenced copy of the gene in the genome.
  • Gene expression differs considerably in different organs, i.e. it is very high in leafs, low in stems and nearly undetectable in flowers.
     The highly regulated promoter of CXc750 offers the possibility to use it for controlled manipulation of transgenic plants. We, therefore, intend to design plant promoters which might serve different needs in genetic engineering of crop plants, such as leaf specific gene expression or controlled production of toxic compounds only upon pathogen infection. This will, most likely, greatly reduce the risk of resistance development in the pathogens.
    Our system provides the tools for a successful accomplishment of our research project.
  • Organ specific gene expression has already been shown.
  • Knowledge, which will derive from analysis if the regulatory sequences of the silenced genes, present in certain A. thaliana ecotypes, offers the possibility to design promoters which have lost the constitutive function of gene activation but retain the ability to respond to pathogen infection.
  • Copies of CXc750 have also been found in the genome of important crop plants, such as Brassica, which indicates that the engineered promoter is likely to function in other plant species.
     It is intended to accomplish this research project in collaboration with Prof. N.J. Panopoulos at the Institute of Molecular Biology and Biotechnology in Heraklion, Greece and possibly Prof. M. Daniels at the Sainsbury Laboratory in Norwich, UK.
    Any interested company for this specific project within the framework of the 4th EU Biotechnology Program is welcome to contact Dr. Charlotte Grimm, phone:(+43)-1-79794-173, fax:(+43)-1-798-7194 or fax:(+43)-1-7979-4131

    Hmc-network: Gene Silencing In Transgenic Plants


  • Peter Meyer, Max-Delbrck-Laboratory, Carl-von-Linné Weg 10, D-50829 Cologne.

    The network was initiated by eight different laboratories located in six European countries to identify molecular mechanisms responsible for the phenomenon of gene silencing in transgenic plants and the molecular analysis of underlying mechanisms and approaches towards stabilisation of transgene activity. Meanwhile, three other European research groups have become affiliated to the network.

    The term 'gene silencing' refers to a complete or partial inactivation of gene activity in transgenic plants. This effect can occur either in primary transformants or during further breeding and propagation of transgenic plants. Transgenes or endogenous genes can also become silenced under the influence of a second homologous copy. The silencing effect can be unidirectional (trans-inactivation) or bidirectional (co-suppression) and it can be influenced by developmental and environmental factors. Inactivation is either associated with a loss of transcription, often corresponding to hypermethylation within the promoter region, or attributed to post-transcriptional degradation of RNA.

    The network concentrates on two major tasks: The establishment of cooperative links between the participants and other European researchers interested in gene silencing and the development of molecular techniques that are necessary to analyse and differentiate among different silencing systems. Therefore the network members organize regular meetings about twice a year to discuss cooperation and to present their latest data to the public. In addition a few external speakers are invited to each meeting. Network meetings haven been held in Cologne, Gent and Basel. The next meeting will be organized in Salzburg in June 95. Technology development currently focuses on RNA turnover, DNA methylation and chromatin structure, in situ hybridisation and developmental regulation of silencing. Individual labs will elaborate the relevant technology and serve as training centres for other participants. Based on this work the network members intend to submit a proposal for the BIOTECH programme of the Commission addressing the mechanisms and regulation of gene silencing in plants. For further information please contact: dr. Peter Meyer, phone:(+49)-221-5062-610, fax:(+49)-221-5062-613 or E-mail: pmeyer@mpiz-koeln.mpg.de

    Activities at CPRO-DLO


  • Dr. Arjen J van Tunen and dr. Tini Colijn-Hooymans, Centre for Plant Breeding and Reproduction Research, P.O.Box 16, NL 6700 AA Wageningen, The Netherlands.
    During the past decades the significance of plants and plant tissue cultures as producers of economically important primary and secondary metabolites has increased dramatically. These compounds include for instance products used in agro-industrial companies such as flavours and fragrances for the food and perfume industry and medicinal compounds for pharmaceutical companies. An important step in the production process is the purification of the compounds which are often only present in low amounts. Therefore universities, institutes and breeding companies invested heavily in Research and Development to improve purification methods and protocols enabling the purification of the desired products in large quantities. However often these strategies are time consuming and economically unattractive.

    To develop an alternative approach which starts at the very beginning of the production process the DLO-Centre for Plant Breeding and Reproduction Research (CPRO-DLO) has initiated a large research project within the department of Cell Biology. In this project a dozen molecular biologists, cell biologists and biochemists aim at using plant cells as factories for a high production of important metabolites. Using a genetic approach we want to select and breed for plants producing high amounts of the desired products. For this, two strategies are followed (see also Figure above):

  • Identification of (exotic) plant species or varieties, specific plant tissues or cells which produce interesting compounds in elevated levels. Therefore data base and gene bank searches in combination with sophisticated cell biological and biochemical experiments are performed.
  • Improvement of the production of metabolites by genetic modification.
    Currently we are using strawberry for the isolation of genes involved in the production of flavours and fragrances and Petunia hybrida and Arabidopsis thaliana for gene isolation and research on flavonoids, steroids and sugar metabolism. Finally fructan biosynthesis is studied in the plant species Helianthus tuberosus. Fructans are fructose polymers which have many potential applications such as their use as low calory sweeteners.

    Given the increasing demand from industry and consumers for natural products and the possibilities plant breeders can offer to increase the production of metabolites in the source plant material, the authors foresee a fruitful future for molecular and genetic breeding approaches. For further information, please contact: Dr. Tini Colijn-Hooymans, fax:(+31)-08370-18094

    Member profiles



    R.A.G.T. Seed Division

    18, rue de S‚guret Saincric

    F-12033, Rodez, France


    R.A.G.T. is a French company, founded by farmers of the Aveyron region, entirely devoted to agriculture since the turn of the century. The abbreviation R.A.G.T. (Rouergue, Auvergne, Gevaudan, Tarn) refers to the original location of the company. At a regional level R.A.G.T. has been a major firm occupying a foremost position in the areas of grain storage, animal and feed production and other supplies for farmers. Today the main business of R.A.G.T., developed through its International Seed Division, is focusing on breeding, production, and marketing varieties of the major agricultural crops adapted to North Western Europe. This activity is concerned with the following plant species: maize, sunflower, forage and turf grasses, small seed legumes, small grain cereals, soy, sorghum and rapeseed. Including its subsidiaries, the R.A.G.T. group employs 850 staff mainly in France but also in Germany, Austria, Holland and Belgium.

    The strong effort devoted to research has placed R.A.G.T. as a leader among plant breeders and seed producers in Europe, especially for corn, sunflower and forage grasses. 20 scientists and 60 coworkers are responsible for the development of new improved varieties. Divided over 8 research centres, located in complementing agroclimatic regions in France, Germany and Holland, most of them are directly involved in traditional breeding and field experimentation. Beside this main expertise, a phytopathology laboratory develops in vitro bioassays, carries out semi natural infestations for the early detection and screening of genetic material resistant to pathogens and biometricians define convenient analysis tools for breeders. Since 1988, a biotechnology laboratory, located at the main breeding station in Rodez, is fully integrated in the research effort. Based on the exploitation of biochemical and molecular markers, in vitro tissue culture, genetic transformation, the challenge of this laboratory is to ensure that the R.A.G.T. breeders have the most advanced tools at their disposal. The activity of the group serves R.A.G.T. by the development of new tools and their application to the breeding programmes in order to:
  • accelerate the breeding process by in vitro culture techniques
  • contribute to a better knowledge of the genetic material through molecular analysis
  • facilitate the screening progress
  • integrate new characteristics in the germplasm by genetic transformation
    R.A.G.T. research activity is reinforced by essential and various collaborations. Thus R.A.G.T. is associated with Dekalb Genetics Corporation (U.S.A.) through a joint venture (G.I.E. Sockalb) in charge of corn, sunflower and sorghum breeding of hybrids adapted to North Western Europe. Through common programmes, on most of the R.A.G.T. crop portfolio, a strongly association exists with the I.N.R.A., the French Research Institute for Agriculture. In the area of biotechnology several projects are carried out in cooperation with French universities and European research groups (e.g. participation in the EU ECLAIR programme). In this context the Plant Industrial Platform is expected to be an instrument for rapid access to new biotechnological products and methods developed in EU funded research programmes. PIP should also offer a chance to meet principal European scientists and certainly contributes to the definition of essential research areas for European plant bioindustry.
    For more information, please contact: Dr. Claude Grand, Société R.A.G.T., 18, rue de Séguret Saincric, 12033 Rodez CEDEX 09, France. phone:(+33)-6573-4100, fax:(+33)-6573-4198

    S&G Seeds

    P.O.Box 26, 1600 AA Enkhuizen,

    The Netherlands


    S&G Seeds is one of the four international seed companies within Sandoz Seeds which, together with Sandoz Pharma and Sandoz Nutrition, form the Life Sciences Sector of Sandoz Limited, based in Basel, Switzerland.

    Sandoz Seeds comprises Roger Seed Co. with its headquarters in Boise, Idaho, USA (vegetables); Vaughan's in Chicago, USA (corn and other field crops); Hilleshog NK in Toulouse, France (sugar beet, corn, sunflower and other field crops) and S&G Seeds in Enkhuizen, the Netherlands (vegetables and flowers).
    With its 4000 employees Sandoz Seeds is active throughout the world in the fields of plant breeding, producing, processing and marketing of seeds and young plants for agriculture, horticulture and floriculture. S&G Seeds, with 1500 employees, concentrates on seeds for vegetables and flowers as well as young ornamental plants which serve as the starting material for professional market gardeners and growers in Europe, Africa and Asia.

    Whereas plant breeding is the core activity in product development, S&G Seeds intensively uses modern technologies to deliver products of the highest quality. Biotechnology is one of the contributing technologies and is carried out in modern facilities located in Enkhuizen. Strategies to characterise and combat plant diseases are being developed. Molecular markers are used to support quality control of seed lots. Crop quality, including health and taste, also forms a significant part of the work being done in Enkhuizen.

    Besides biotechnology, seed technologies are used to optimise product performance. Seed coatings, including biological seed coatings, are being developed in order to decrease the use of pesticides by the grower. Last but not least, research on seed quality and young plant vigour is also part of the technology support in product development.

    S&G Seeds has actively participated in a number of EU projects and has the intention to continue this involvement. It considers industrial involvement in defining the goals and potential applications of EU-funded projects essential. S&G Seeds views the Plant Industrial Platform as a significant instrument in these efforts and also views the platform as a vehicle to disseminate results from projects and to stimulate interactions among industries to define scientific needs for product development.
    For more information please contact Dr. Andre W. Schram, S&G Seeds, P.O.Box 26, 1600 AA Enkhuizen, The Netherlands, phone:(+31)-2280-66178, fax:(+31)-2280-17144

    ACTIVITIES


    Meetings


  • PTP Theme A1 group is convening 1-3rd March in Koln to discuss progress and future developments. PIP members are invited to attend and may contact the PTP programme manager: dr. A. Beadle, JIC, Norwich Research Park, Colney, Norwich, NR4 7UL, U.K., phone:(44)-603-452-571, or fax:(+44)-603-456-844.
  • PTP Theme group B2 meets in Perpignan, France from 16-19th February. PIP members should contact the PTP programme manager, for the address see above.
  • The 13th International Plant Protection Congress will be held in the Hague, the Netherlands fro 2-7th July. The organising secretariat is the Holland Organising Centre, Lange Voorhout 16, 2514 EE the Hague, the Netherlands, phone:(+31)-70-365-7850, or fax:(+31)-70-361-4846

    The PIP Steering Committee


    The composition of the PIP Steering Committee changes at regular intervals, in order to closely involve all PIP members over time with its activities. The current composition is as follows:

    Dr. Leen Davidse, interim chairman
    Dr. Andre Schram (S&G Seeds), finances
    Dr. Simon Bright (Zeneca Seeds)
    Dr. Reinhard Nehls (Planta / KWS)
    Dr. George Freyssinet (Rhone Poulenc)