ABSTRACT

The objective of "ZEASTAR" is to use the powerful tools of plant genomics to generate a functional blueprint for the Zea mays endosperm cell factory. To do this, the 10 partners have identified five key stages in endosperm development, which are all of considerable interest to both European academic laboratories and plant breeding companies. We intend to characterise each of these five stages in endosperm development, using procedures, which will be developed jointly by the partners.

If we are successful we will produce a functional blueprint for endosperm development, identify useful variations and develop technologies and material which will find immediate application in the characterisation of several biochemical pathways, which are currently being targeted by all plant breeding companies within the EU.

OBJECTIVES

The aim of our programmeme is to meet in full the objectives of the Cell Factory.

Key Action, so that new knowledge will be generated on one of the most efficient cell factories known to western agriculture; the maize endosperm, through genomics, proteomics, high-throughput screening and biochemical engineering. Scientifically, the consortium will develop and use high-density EST arrays, gel-based proteomics and gene knock-outs. These tools will enable the consortium to identify and characterise the key genes involved in grain development. By producing this information (consisting of transcriptome maps, proteome maps, specific gene knock-outs and the biochemical characterisation of endosperms), we will put European scientists in a strong position to compete with the currently-dominant US agri-biotechnology giants.

DESCRIPTION OF THE WORK

This proposal will use the 10 partners' (6 academics' and 4 companies') considerable experience and various technologies to produce part-normalised cDNA libraries from five key stages in endosperm development. Unique ESTs isolated from these libraries will be used to construct high-density EST insert arrays. These arrays will be used to characterise the expression of up to 7,500 endosperm genes in endosperm development, via state-of-the-art bioinformatics. EST array technology will be coupled with 2-D gel electrophoresis to profile the developing endosperm's proteome.

The results from these complementary studies will be directly compared. ESTs and proteins identified as being of interest, (for instance those having a highly specific temporal and spatial expression profile) will be funnelled into the programme's various gene machines to produce specific gene knock-outs.

In turn these knock-outs will be characterised for modified endosperm traits by:

• The high density EST arrays.
• 2-D gel electrophoresis and proteomic technology.
• The measurement of key biochemical parameters.

Characterisation of the endosperm to this high degree will provide both further research materials for the academics laboratories involved and material for the plant breeders and food processors to include in their respective pipelines. It is likely that both the characterisation and use of this material will continue beyond the lifetime of this programmeme. To achieve our goals and to keep the programme highly focussed, the work will be organised into five separate work packages, with each work package having its own co-ordinator, objectives and optimal combination of participants.

DELIVERABLES

• Consortium Agreement.
• 20 kernel specific cDNA libraries.
• Sequence of 20,000 ESTs.
• Unique set of 7,500 kernel ESTs.
• High-density colony protein expression arrays.
• First Year Report to the Commission.
• Standard array format.
• Standard arrays distributed to parties.
• Expression data on 7,500 kernel EST.
• Standard 2-D protocol for maize kernel.
• 1Second Year Report to the Commission.
• 1Proteomics data on kernel development.
• 1Protein sequence for selected samples.
• 1Comparative information on EST and protein expression.
• 1~50 kernel specific gene knock-outs.
• 1Standard protocol for characterisation of maize kernels (normal and mutant).
• 1Biochemical/Physiological data on maize kernels (normal and mutant).
• 12-D gel database of normal and mutant endosperms.
• 1Rational data base linking gene expression, the proteome and biochemical data.
• 20. Third Year and Final Report to the Commission.

CONSORTIUM