MADS box transcription factors appear to be the key regulators of plant development and play pivotal roles in the timing of flowering, the initiation of floral organs, fruit development, and pod-shattering. Genetic and molecular analyses revealed that these proteins are active in a combinatorial manner, and over the last decade the yeast two-hybrid system has been used to identify dimerization among plant MADS box transcription factors. Although this is a high-throughput and fast screenings method, false positive interactions are frequently identified and therefore this system could often not be used for transcription factor analysis because of technical restrictions (auto-activation). Furthermore, the two-hybrid system gives a statically visualized view of interactions. It shows not only that two proteins are able to physically interact, but also that inside a living plant, cell interactions are dynamic, proteins are moving, and there is a competition for various interaction-partners and some transient interactions are stabilized by a third protein. Therefore, there was a clear need for a technology that could enable researchers to identify protein-protein interactions invivo. In this paper a new method is described and applied that enables us to follow the proteins in living plant cells and to detect specific interactions. It showed for the first time specific interactions inside living plant cells and the transport of MADS box transcription factors from the cytoplasm into the nucleus upon dimerization.

This paper describes the usage of Fluorescence Resonance Energy Transfer (FRET) microscopy for the detection of protein-protein interactions invivo. Detection of FRET in plant cells is not easy because of the high levels of auto-fluorescence by chlorophyll. Therefore, both spectral imaging (SPIM) and Fluorescence Lifetime Imaging (FLIM) were used to detect FRET. This latter method is very sensitive and independent of variations in expression level of the fluorescently-tagged proteins. Dr. Dorus Gadella has implemented the FRET-FLIM technology for usage in plant cells at the MicroSpectroscopy Centre of Wageningen University. Currently, the system is used routinely for the detection of FRET and the technology has been further fine-tuned. In addition many other technologies have been developed, such as FCCS (Fluorescence Cross-Correlation Spectroscopy) that enables us to study protein-complex formation and protein dynamics inside living cells.

Many complete genomes have been sequenced and at this moment one of the greatest scientific challenges is to identify the functions of all genes along with their encoded proteins. Almost all proteins appear to be active in complexes and hence the knowledge about protein-protein interactions is very important in order to gain insights into the functioning of these proteins. The technology used by our group can be broadly applied to fit all types of proteins existing in all kinds of organisms. The work on the plant MADS box transcription factor family is of the greatest importance in our understanding of plant development. The formation of floral organs and fruits appears to be initiated by these genes and hence knowledge about their molecular working mechanisms will help to shed light on the regulatory pathways guiding plant development.

Our group, under the leadership of Professor Gerco C. Angenent, has been involved in plant MADS box transcription factor research for many years. The research described in this paper was a part of my Ph.D. project. We performed various yeast two-, three-, and four-hybrid screens and were looking for suitable methods to confirm the identified interactions by an alternative technology and one which would preferably be found within their natural environment. The use of FRET-FLIM technology appeared to be a very reliable and suitable method for that purpose.