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Final Activity Report Summary - ANALYSIS OF VRN5 (Isolation of the VRN5 gene and the molecular characterization of its function in the vernalization response of Arabidopsis thaliana)

To avoid flowering before the unfavourable conditions of winter many plant species of the temperate regions have to experience several weeks of cold temperatures before the floral transition can occur. The acceleration of flowering by long periods of cold is called vernalisation and is extensively studied at the molecular level in late flowering ecotypes of Arabidopsis thaliana. A major effect of vernalisation is the gradual down regulation of the floral repressor gene FLC. It was shown that this effect is mediated by the function of several VRN/VIN genes which are involved in the transfer of FLC chromatin into a condensed state where gene transcription is stably repressed.

The vrn5 mutant represents a new component of the vernalisation pathway, whose isolation and characterization were the main objectives of the project. The seven available vrn5 mutant alleles show a clear reduction in their vernalisation response which correlates with a high level of FLC expression after vernalisation. As a first step towards its characterisation, the VRN5 gene was isolated by a map-based cloning approach. VRN5 (gene number At3g24440) encodes a protein of 602 residues carrying a FNIII domain and a PHD finger motif and belongs, together with VIN3 (1), to the VEL (Vernalisation-like) gene family in Arabidopsis (2).

GUS staining of plants transformed with a VRN5-GUS reporter construct, which was able to complement the vrn5-1 mutation, revealed VRN5 expression in the apical region of the shoot, in the vasculature of the hypocotyl and root, and in the root tip. RT-PCR experiments showed that VRN5, in contrast to VIN3 whose expression is induced by long periods of cold, is constitutively expressed independent from temperature. Stable expression of a functional fusion of the VRN5 protein with the fluorescent EYFP protein under the control of the VRN5 promotor in Arabidopsis plants, showed VRN5 to be localized to the nucleus, under all conditions tested. Taken together, these results suggest that the vernalisation dependent activity of the VRN5 protein is not regulated by its expression levels or its localisation, but is regulated by a modification of the protein activity itself.

To test whether the activity of VRN5 is regulated by a physical interaction with other proteins of the vernalisation pathway Yeast-Two-Hybrid experiments were performed. These experiments, as well as independent in vitro analyses, showed that VRN5 and VIN3 are able to form heterodimers in plants mediated by their C-terminal VEL domains. Furthermore, transient co-expression of fluorescence labelled versions of both proteins in Arabidopsis leaf cells revealed the co-localization of VRN5 and VIN3 in nuclear speckles. ChIP experiments, done in collaboration with Dr. Pedro Crevillen, showed that in vrn5, similar as it is described for vin3, vernalisation induced deacetylation of histone tails at the FLC locus is not detectable. Taken together, the results suggest a concerted function of VRN5 and VIN3 by the formation of a heterodimer as soon as VIN3 expression is induced during long periods of cold temperatures.

Furthermore, extensive double mutant analyses between all currently available vernalisation (vrn/vin) mutants argue for the existence of two separate branches of the vernalisation pathway, both involving VRN5.

In summary, the scientific work done in this project led to a more detailed understanding of the molecular basis of vernalisation by the identification of a new component and uncovering its relationship to other factors of the pathway. As a result of this, it opens up the possibility to reveal the role of homologous genes in the vernalisation response pathways of relevant crop species, an approach which is currently underway at the John Innes Centre in Norwich.

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