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Introduction
The maize kernel is not merely a seed but a one-seeded fruit, in which the seed, consisting of embryo and endosperm, is permanently enclosed in an adhering pericarp. Of the various kernel components, the endosperm (which consists mostly of starch) constitutes about 85% of the kernel dry weight, the embryo 10% and the pericarp and seed coat the remaining 5%. Virtually all of the starch within the endosperm is synthesised within a 25 day period from day 10 to about day 35 post fertilisation. During this time the endosperm increases in size from less than 0.1mm to up to 10mm, a figure which represents a volume increase of about 1400 fold. Not all maize endosperms have the same constitution, and different types of maize are used variously in food, animal feed and industrial products.

Each year in Europe approximately 10,969,000 hectares are planted with hybrid maize seed which results in ~11% of world maize production. 57% of the EUs industrial (non-food) starch market is obtained from maize, whilst 71% of starches for food purposes are also derived from maize. Despite the utility and value of maize, the crop can still be improved in terms of nutrition, energy content, kernel and starch types, and yield under European conditions. Because of this and the expanding market for maize-derived products, European-based plant breeding companies have initiated breeding programmes (using modern molecular breeding techniques) designed to generate elite material specifically for European conditions. It is the aim of this programme to support this company-based work and carry out further basic and applied research to further enhance our knowledge of the maize kernel and its contribution to endosperm development.

ZEASTAR is funded by the European Union (EU), Award No QLRT-2000-00020: The Cell Factory, Section 3.3.2 The project is coordinated by Prof. Keith Edwards, School of Biological Sciences, University of Bristol, Long Ashton, Bristol BS41 9AF. Email: keith.edwards@bbsrc.ac.uk

The ZEASTAR partners are:

1. Keith J. Edwards, University of Bristol, Email: keith.edwards@bbsrc.ac.uk
2. Alain Murigneux, Rhobio/Biogemma, Email: alain.murigneux@biogemma.com
3. Simon Bright, Syngenta Wheat Improvement Centre, Email: simon.bright@syngenta.com
4. Mario Motto, Istituto Sperimentale per la Cerealicoltura, Email: motto@tin.it
5. Richard D. Thompson, INRA-URGAP Legume Unit, Email: thompson@epoisses.inra.fr
6. Michael Burrell, Advanced Technologies (Cambridge) Ltd., Email: mmb.atc@dial.pipex.com
7. Milena Ouzunova, KWS SAAT AG, Email: m.ouzunova@kws.de
8. Klaus Palme, Max-Planck-Gesellschaft, Email: palme@mpiz-koeln.mpg.de
9. Catherine Damerval, INRA SGV, Email: damerval@moulon.inra.fr
10. Jean-Louis Prioul, Institut de Biotechnologie des Plantes, Email: jean-louis.prioul@ibp.u-psud.fr

Maize Kernel Development

Figure 1 Primary endosperm cell in vitro 1 day after fertilisation. From Krantz et al. 1998	Stage 1  0 to 96 Hours Post Fertilisation: Nuclear Division and Cellularisation of the Coenocyte Double fertilisation of the egg cell and the central cell in the embryo sac results in formation of the diploid embryo and the triploid endosperm (figure 1) respectively. The endosperm nucleus divides synchronously over the first 72 hours without producing cell walls, resulting in a coenocyte of up to 1000 nuclei. This occurs in the peripheral cytoplasm of the embryo sac, and surrounds a large central vacuole. Cell wall formation then separates the nuclei from one another, as in figure 2.
Figure 2 In vitro endosperm after 4 days in culture. From Krantz et al 1998	Stage 2 4-10 Days: Cell Division and Differentiation The separated nuclei again divide, but this time in a more orderly anticlinal fashion, to give an inner layer of nuclei which will give rise to the central endosperm initials, and an outer layer which will form the aleurone initials. Division of the endosperm cells continues until the endosperm cavity is filled, giving a final population of up to 200,000 cells (figure 3). The aleurone initials divide anticlinally to give a single cell outer layer to the endosperm cavity. The basal endosperm cell layer becomes morphologically distinct from the rest of the aleurone, characterised by modified cell walls and vesicle-rich cytoplasm.
Figure 3 A Maize kernel	Stage 3 12 Days to Maturity: Reserve Synthesis During this period the endosperm accumulates large amounts of starch and storage proteins. The basal endosperm transfer cells are responsible for the uptake of assimilates into the endosperm during the main period of grain filling. This phase of endosperm development is also characterised by the occurrence of endoreduplication in the central endosperm cells. DNA replication occurs in the absence of mitosis or cytokinesis, resulting in large nuclei with DNA contents of 16C or higher. This has been suggested as a mechanism to enhance the synthesis of storage proteins by increasing the supply of DNA template.
Figure 4 Mature maize cobs	Stage 4 Dry Down At around 25-30 days after pollination, the relative water content of the endosperm begins to decrease. The initiation of seed desiccation provides a signal to arrest germinative development. The embryo and aleurone accumulate gene products associated with desiccation tolerance. The central endosperm cells do not accumulate these products, and are inviable on seed germination.
Figure 5 A germinating maize shoot	Stage 5 Germination Following imbibition of the kernel, a battery of hydrolytic enzymes are induced by a gibberellin hormone signal in the aleurone, and to a lesser extent in the scutellar epithelium of the embryo. These enzymes break down the starch and storage protein reserves in the central endosperm, producing the nutrients required by the embryo for germination and initial growth before photosynthesis take place. The triacylglycerols stored in the embryo are also broken down during this period.

ZEASTAR Objectives
ZEASTAR will examine in considerable detail the transcriptome and proteome of the developing maize endosperm. This information will be used to target distinctive, previously uncharacterised endosperm specific genes which will be knocked out via Mutator transposon tagging. Normal and mutant plants will be examined for modified endosperm characteristics. Lines with either beneficial or unusual characteristics will be included in the industrial partners breeding programmes for possible use as a source of novel endosperm products.

To fully understand the processes that lead to endosperm development, ZEASTAR believes that it will also be necessary to examine the parallel processes that occur (simultaneously and through crosstalk) within the surrounding kernel. To do this, the 10 partners will utilise their considerable experience and various advanced technologies to produce 20 maize cDNA libraries from five key stages in both endosperm and kernel development. 20,000 of these ESTs (1,000 from each library) will be single pass sequenced. 7,500 unique ESTs will be used to construct high density EST insert arrays. These arrays will be used to characterise the expression profile of the maize endosperm via state of the art bioinformatics.

In addition, the consortium will utilise 2-D gel electrophoresis to profile the developing kernel’s proteome. The results from these complementary studies will be directly compared to produce a functional blueprint for endosperm development. ESTs and/or proteins identified as being of interest (for instance those having a highly specific temporal and spatial expression profile) will be funnelled into the programmes various gene machines to produce specific gene knockouts. In turn these knockouts will be characterised for modified kernel or endosperm traits by:

1. High density EST technology.
2. 2-D gel electrophoresis and related proteomics technology.
3. Measurement of several key kernel biochemical parameters.

Characterisation of the normal and modified endosperm will provide both further research material for the academic libraries involved, and material for the plant breeders and food processors to include in their respective research or product pipelines. It is likely that both the characterisation and utilisation of this material will continue beyond the lifetime of this programme.

ZEASTAR Achievements so far
Generation of cDNA libraries from developing endosperm and kernel. Using material generated in Bergamo, Italy, the partners have generated all of the 20 libraries described in the original proposal. In addition, extensive sequencing of the various libraries has been carried out. In most cases 1,000 clones have been sequenced from each library. The sequences and clones have been assembled in order to design a unigene set. This unigene set will be distributed to the appropriate partners sometime during April 2002.

Generation, characterisation and utilisation of high-density EST arrays This will begin once the ZEASTAR unigene set has been collated. However, several of the ZEASTAR partners have already begun to develop microarray-based technologies.

Characterisation of the developing kernel and endosperm via 2-D electrophoresis 2-D electrophoresis has now been successfully optimised for studying both maize kernels and endosperms. The technology is new well placed for exploitation by the ZEASTAR members.

Generation of endosperm specific gene knock outs Two partners have gene knock out technology. Within the ZEASTAR programme one of these has started to generate gene knock outs for maize genes identified by the ZEASTAR partners.

Functional characterisation of gene knock outs During the first 12 months of the ZEASTAR programme three partners have developed and implemented the required biochemical technologies to characterise both the F2 developing grain and the various gene knock outs.

Maintained by Gary Barker Last updated May 2002