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Establishing an integrated network model for secondary wall thickening in anther development.

Reference	BB/F021062/1
Principal Investigator / Supervisor	Professor Zoe Wilson
Co-Investigators / Co-Supervisors	Professor Andrew Wood
Institution	University of Nottingham
Department	Sch of Biosciences
Funding type	Research
Value (£)	502,722
Status	Completed
Type	Research Grant
Start date	18/07/2008
End date	17/11/2011
Duration	40 months

Abstract

Secondary plant cell wall thickening is vital for many aspects of plant growth, typically for the production of tissues for water transport and support, but also when mechanical force is required. Secondary thickening occurs in the anther endothecium and is vital for the physical forces needed for anther opening, as demonstrated by our Arabidopsis non-dehiscent ms35 mutant, which prevents secondary thickening in the anther endothecium, while other tissues remain normal. The ms35 mutation is due to a defect in the MYB26 gene, which regulates secondary thickening and can ectopically induce thickening. We aim to determine the nature of activation of the MYB26 protein complex and reveal, and functionally test, the MYB26 regulatory network controlling secondary thickening in a multi-disciplined programme using systems biology resources available through MyCIB and CPIB. We have identified a zinc finger family protein, which we propose interacts with and activates MYB26. We will determine its expression pattern using fluorescently tagged fusion proteins, confirm the interaction in vivo with MYB26 by FRET/FLIM or BiFC analysis and affinity purification of the protein complex, and determine its effect on activation of MYB26. We aim to establish the regulatory network for MYB26. Firstly by testing our current model, that MYB26 directly regulates NST1 and NST2 to bring about secondary thickening. This will be done by EMSA and ChIP analyses. We will then use an inducible MYB26 construct and QPCR to generate a time-series analysis of expression profiles for genes previously shown to have altered expression in the ms35 mutant. Mathematical and statistical analyses will be used to develop a dynamic predictive model for this network. This model will be used to predict key components in the pathway. The network topology and functional effects of these will be tested in KO and OEx lines by QPCR, FTIR, monosaccharide analysis and immunolocalisation.

Summary

Secondary plant cell wall thickening is vital for many aspects of plant growth, typically for the production of mechanical tissues for water transport and support, but is also critical when other aspects of mechanical force are required, for example anther dehiscence. Secondary cell walls are composed of cellulose, hemicellulose and lignin. Advances have been made towards understanding the biosynthesis of these wall components, however little is known about the factors that control these pathways. Such regulatory networks tend to initiate the biosynthesis of multiple wall components, dissecting these regulatory networks may permit the separation of these pathways with the future goal of controlling specific components of the cell wall matrix in isolation from other components. Understanding such processes is vital for the manipulation of secondary thickening for modification of wood quality, biofuels, but also for the manipulation of processes requiring such mechanical forces, for example anther dehiscence and the control of male fertility for hybrid development and control of gene flow. Secondary thickening occurs in the anther endothecium and is vital for the physical forces needed for anther opening, as demonstrated by our Arabidopsis male sterile mutant ms35 which produces normal pollen but is male sterile because the pollen is not released. We have shown that this defect is caused by a mutation in MYB26, a gene that has homology to members of the MYB family of transcriptional regulators. We have shown that over-expression of MYB26 can switch on secondary thickening in tissues that do not normally produce secondary thickening (ectopic secondary thickening). In these over-expressing lines we see increases in the expression of a number of genes, including transcription factors, but also biosynthetic components of the secondary thickening pathway. We have constructed a preliminary model for this pathway and hypothesise that two NAC domain genes may be direct targets for MYB26. We have also identified a large number of genes that have not yet been placed into this pathway. We propose to test our current model and determine the primary regulatory targets for MYB26, but also to extend our model to include the other genes that have altered expression in our mutant. We will do this by carrying out a time-course analysis of these genes after MYB26 is switched on. This will identify those genes that are direct targets of MYB26 which are switched on very soon after MYB26 expression, but also those genes that are switched on later in the pathway, possibly by factors other than MYB26. We will use this information in collaboration with mathematicians/systems biologists to develop an integrated model for this pathway. We will then test this model by selecting genes that are predicted to be key regulatory points in the pathway and investigate the effects of knocking-out (mutating) or over-expressing them. We will check the order of our network by analysing the expression of down and up-stream genes in these backgrounds and determine the effect on gene expression of subsequently expressing MYB26 in these backgrounds. We will also establish the functional effects of these genes by investigating changes to the structure of the cell wall in the knockout, or overexpression lines. We have also found a protein that appears to binds to MYB26 and may serve to activate it to enable it to carry out its normal function. We will test where this protein is expressed and whether it activates the MYB26 protein. This will provide valuable information on the first step in the MYB26 pathway in determining the activity of MYB26.

Committee	Closed Committee - Plant & Microbial Sciences (PMS)
Research Topics	Plant Science, Systems Biology
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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