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Characterisation of Plant MADS Box Transcription Factor Protein-protein Interactions
Richard G. H. Immink
其他書名
Use of Petunia Hybrida as a Model System
出版Wageningen Universiteit, 2002*
ISBN90580863999789058086396
URLhttp://books.google.com.hk/books?id=fITLAAAACAAJ&hl=&source=gbs_api
註釋To determine whether dominant-negative mutations can be used more generally to obtain MADS box transcription factor mutants, several mutated forms of PFG were overexpressed in petunia. In addition mutations for the MADS box transcription factor APETALA1 (AP1) from Arabidopsis were generated and tested for their ability to repress AP1 gene function in a dominant-negative manner. Unfortunately, overexpression of none of the generated constructs resulted in a dominant-negative effect, demonstrating that it is difficult to generate a universal dominant-negative strategy. Nevertheless, this study has elucidatedrevealed the importance of some motifs and domains in the AP1 protein for its functioning. From these experiments it was also clear that more knowledge about the function of specific MADS box protein domains and their interactions is essential to predict which domain needs to be modified to obtain a dominant-negative version. To obtain information about the interactions between MADS box transcription factors, yeast two hybrid-experiments can be performed, because these systems have proven to be powerful methods to determinegain knowledge about protein-protein interactions. However, no knowledge is obtained about the occurrence existence of these interactions in living plant cells and in which cell compartments the interactions take place. To get insight in the biological relevance of the identified interactions, in-planta analyses need to be performed. For this reason a few of the identified petunia MADS box protein dimers were analysed in leaf protoplasts by means of spectroscopy techniques based on Fluorescence Resonance Energy Transfer (FRET). All the in yeast identified heterodimers for the ovule specific MADS box protein FBP11 could be confirmed in living cells and, in addition, homodimerisation was observedtained for the MADS box proteins FBP2, FBP5 and FBP9. Subsequently, localisation studies were conductedperformed with full-length and truncated proteins lacking the supposed bipartite Nuclear Localisation Signal (NLS), making use of Confocal Laser Scanning Microscopy (CLSM). The obtained results demonstratedshowed that both partners of a MADS box protein dimer need to contain the NLS signal for nuclear localisation and furthermore, dimerisation appeared to be essential for this translocation from cytoplasm to nucleus. All these observations demonstrate again the importance of protein-protein interactions for MADS box protein functioning. The knowledge about these interactions does not only teach us about the molecular mechanisms underlying MADS box transcription factor functioning but gives also information about the presence of functional redundancy among members of this large family of transcription factors. Furthermore, a pilot experiment has shown that protein-protein interaction screenings can be exploited for the identification of functional homologues (orthologues) of specific MADS box proteins from species that are genetically not very well characterised. These findings demonstrate that interaction mapping provide an additional tool for functional genomics.