Using STJs to measure the expression and interactions of Arabidopsis fluorescently-labelled proteins involved in pollen development

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Pollen development is a critical step in the plant life cycle with commercial importance in crop breeding and seed production. We have been investigating the role that the MS1 protein, which is vital for pollen formation, plays in this process. We have identified a number of proteins that MS1 appears to interact with, but need to verify these interactions in planta. To do this we need to show when and where these proteins are expressed and confirm that they interact with MS1. One way to do this is to make fusion proteins combining a fluorescent marker to the protein of interest and to look for fluorescence and whether there is a change in the fluorescence as a consequence of interacting with the MS1 protein labelled with a different fluorescent marker. Detection of fluorescently labelled proteins are usually conducted by confocal microscopy, however, this is very difficult as the proteins are expressed in a limited number of cells within a tissue, chlorophyll autofluorescence partly masks the fusion protein fluorescence, and detecting small changes in fluorescence is extremely difficult. Although these issues apply to our work they are also common limitations encountered by other Plant Science researchers. One approach that may alleviate many of these problems is to use a novel form of detector (STJ) that detects the energy of individual photons. STJs for optical wavelengths have been developed by the European Space Agency and applied to the detection of biological fluorescence at the University of Leicester Space Research Centre. STJs measure the energies of individual optical photons with high efficiency and extremely low internal background. The detector offers an advantage over other photon counting systems because the entire fluorescent spectrum is detected on an event-by-event basis. This means that the contributions of different fluorescent labels can be distinguished without the need for filters and problems of autofluorescence can be eliminated. We wish to apply this technique to the detection and interaction of fluorescently labelled proteins in the anthers of a higher plant. This will validate this technique for the study of in vitro expression and interaction of proteins in plants. The use of STJ-based detection systems in plant biology may overcome many of the limitations that are currently encountered in the visualisation of fluorescent molecules in planta. This work will also provide valuable information on the process of pollen development.