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Reverse genetic analysis of plant secondary cell wall biosynthesis and function

Reference	BB/C505632/1
Principal Investigator / Supervisor	Professor Simon Turner
Co-Investigators / Co-Supervisors	Professor Royston Goodacre
Institution	The University of Manchester
Department	Life Sciences
Funding type	Research
Value (£)	297,321
Status	Completed
Type	Research Grant
Start date	01/03/2005
End date	29/02/2008
Duration	36 months

Abstract

Using a forward genetic screen mutants in both cellulose and lignin formation have been identified based on their irregular xylem (irx) phenotype. These mutants have answered important questions about the organisation, localisation and assembly of the cellulose synthase complex. Despite this progress no genes involved in xylan biosynthesis have been identified even though it is one of the most abundant biopolymers. It is also unclear what other components may be essential for secondary cell wall (SCW) function. This and other data suggest forward genetics has not identified a complete list of all the genes required for SCW formation. An alternative approach is to use reverse genetics. Current understanding of plant metabolism is epitomised by the lignin biosynthesis pathway. This pathway has been re-written several times in the last 10 years. Much of the confusion was caused by the presence of conjugated precursors that were not amenable to conventional phenylpropanoid analysis. It is likely that the phenylopropanoid pathway is representative of much of plant metabolism and gives some indication of the challenges required to obtain an accurate picture of plant metabolism. In contrast to targeted metabolic analysis, methods such as NMR or Fourier Transform InfraRed spectroscopy (FTIR) use relatively unbiased extraction methods to generate a fingerprint that is representative of most, if not all, of plant metabolism. A combination of gene expression data, reverse genetics, unbiased measurement of metabolic intermediates together with proper analysis offers an unprecedented opportunity to re-examine plant metabolism. A pilot study has used microarray analysis to identify genes that are co-regulated with IRX3 a known marker for SCW synthesis. The list identifies other known SCW markers such as irx1 and inx5. More importantly, reverse genetic analysis has confirmed that this method has identified at least four novel genes that are essential for SCW formation. This proposal aims to extend the reverse genetic study to a larger number of genes. Methods will be developed to rapidly fingerprint SCWs and metabolites using FT-IR, and possibly also NMR. A training set composed of known SCW mutants that have well characterised defects in cellulose and lignin biosynthesis as well as the cytoskeleton mutants will be used in conjunction with supervised analysis methods to determine the optimal method for discriminating between the different mutant classes. This method will be used to categorise novel mutants and assign them to a particular biosynthetic pathway and to identify a complete set of genes required for the cellulose and xylan biosynthesis pathways. Further detailed characterisation will be performed to assign these genes a particular function within the pathways. Assigning gene function represents both a challenging and exciting process. Several of the genes co-expressed with IRX3 exhibit no homology to any known genes. This is representative of the Arabidopsis genome as a whole where a large proportion of the genes share no homology with genes of known function. Assigning a function to these genes remains one of the biggest challenges in the post-genomic era. There are several important advantages for using SCW synthesis for metabolomic and expression studies. It is the predominant metabolic process during certain stages of stem and hypocotyls development, there are several known markers genes that highly and specifically expressed during SCQ formation and finally it is possible to examine both the metabolic intermediates (metabolome) and obtain a sensitive fingerprint of the final product (the cell wall) both of which may be used to give crucial information about gene function. The information gained from studying a tractable system such as SCW biosynthesis is likely to be applicable to many other aspects of plant metabolism.

Summary

unavailable

Committee	Closed Committee - Plant & Microbial Sciences (PMS)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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