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Role of VRN5/VIN3 in the vernalization response of arabidopsis

Reference	BB/C517633/1
Principal Investigator / Supervisor	Professor Dame Caroline Dean
Co-Investigators / Co-Supervisors	Dr Joshua Mylne
Institution	John Innes Centre
Department	Cell and Develop Biology
Funding type	Research
Value (£)	227,193
Status	Completed
Type	Research Grant
Start date	01/02/2005
End date	31/07/2008
Duration	42 months

Abstract

A central objective of the Dean group is to understand the molecular basis of vernalisation in Arabidopsis. Central to vernalisation is the regulation of a gene called FLC. Previous work has established that prolonged cold represses expression of FLC and this repression is maintained even when plants are returned to warm conditions. Maintenance of this repression is mediated by a set of Arabidopsis genes called VRN genes. This proposal will build upon the recent cloning of two related genes (VIN3/VRN7 and VRN5). The proteins contain PHD motifs that have been shown to function as nuclear phosphoinositide receptors in human cells. The expression of one of the proteins, VIN3, is induced by prolonged cold whereas RNA analysis of VRN5 suggests it is present at similar levels before, during and after the cold. However, there is evidence for alternative transcript processing of both genes. Yeast two hybrid analysis has shown the two proteins strongly interact. The model to be tested is that VIN3 and VRN5 protein expression is cold-regulated and their function is activated by a cold-induced phosphoinositide signalling pathway. The activated heterodimer would mediate histone deacetylation changes at the FLC locus and this would initiate a sequence of events at FLC involving histone methylation and recruitment of silencing complexes. Experiments aimed at characterising VIN3 and VRN5 transcript processing by northern analysis and RT-PCR will be undertaken. Expression of VRN5 protein will be monitored by production of a polyclonal antibody and western analysis and construction and monitoring of a translational VRN5-GUS fusion. In a collaboration with Dr Divecha (Amsterdam) the role of the PHD fingers will be analysed by generating the proteins in E. coli and testing for their ability to bind phosphoinositides. In vivo relevance of any potential binding will be tested by site-directed mutagenesis to reduce phosphoinositide binding and then testing the ability of the protein to complement a loss-of-function vrn mutation. The in vivo interaction of VIN3 and VRN5 will be assayed by co-immunoprecipitation using transient transfection experiments in Nicotiana benthamiana and also using stable Arabidopsis transgenics expressing tagged versions of the proteins. Other proteins interacting with VIN3 and VRN5 will be identified using a yeast two-hybrid analysis (through collaboration with Dr Shin-ichiro Kidou) and their role in vernalisation investigated through analysis of T-DNA insertion mutants. Genetic analysis will be used to define the epistatic relationships of vin3, vrn5, vrn2 and vrn1. If VIN3 and VRN5 function simply as a heterodimer with a common function then the vin3 vrn7 and vrn5 double mutant will not give an additive phenotype. In addition, to assess if VRN2 VRN1 function follows that of VIN3 VRN5 we will generate and analyse double mutants between vrn5 and vin3 vrn7 mutants with mutations in VRN1 and VRN2. lastly, ChIP experiments will be undertaken to determine if vrn5 and mutants generated from T-DNA insertions in VIN3 and VRN5 interactors affect the FLC cold-induced histone changes.

Summary

unavailable

Committee	Closed Committee - Genes & Developmental Biology (GDB)
Research Topics	Plant Science
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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