Project 5 - Coordination of Gene Expression and Enzyme Activity during Growth of Young Seedlings

1. Planned Outcomes/Aims

The overall aim of this project is to define the control mechanisms and coordination of individual genes and biosynthetic enzymes in growing cereal cells, namely in elongating coleoptiles of barley.  Elongating coleoptiles provide a relatively simple biological system in which expansion is the main cellular activity.

2. Contracted Outputs

Specific outputs anticipated for this Project are:

• Generation of EST libraries from elongating coleoptiles
  
  
• Identification of candidate genes for the synthesis and regulation of wall polysaccharides
  
  
• Definition of amino acid sequences of major and minor proteins present in the growing cells
  
  
• Mapped the important genes on the barley genome.

3. Research Methodology

• Microarrays:  We will use high density cDNA arrays, reverse northerns, EST and SAGE libraries to study differences in gene expression between an extending coleoptile/hypocotyl and a fully expanded internode segment, and to identify candidate genes for synthases in cases where no sequence is available (eg. pectin and heteroxylan synthases).  We will produce microarrays of barley ESTs through our collaborations with Dr Andreas Graner at IPK, Gartesleben, Germany and through the International Triticeae EST Cooperative.
  
  
• Proteomics:  Which proteins expressed by coleoptiles/hypocotyls are related to identified genes?  Two-dimensional gel electrophoresis and mass spectrometric sequencing will be used to obtain short stretches of amino acid sequence from proteins located in the Golgi, plasma-membrane or cell wall.
  
  
• Microscopy:  Proteins produced in heterologous expression systems will be used for antibody production; these will be used in immuno-fluorescence and -electron microscopy experiments to study the spatial and temporal coordination of expression of key enzymes or proteins.  In situ hybridization will be used to identify the cells and tissues in the growing plant that express a particular gene of interest.
  
  
• Mapping:  Candidate genes that come out of the gene identification program will be mapped to the barley genome, where co-location with significant QTLs for cell wall components or structure will provide evidence of possible function.

In the course of these experiments we will correlate the expression of major genes for wall synthesis with growth and associated changes in cell wall structure, we will identify minor and regulatory genes involved in wall structure, and will analyse genes linked with cell wall markers.  A transposon-tagged mutant population of barley is being produced by the applicants in collaboration with the group of Professor Horst Lörz in Germany.  We will also characterize existing cereal mutants.

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