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

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.

MS1 is vital for pollen development and regulates transcription in the anther. We have identified, by Yeast 2-Hybrid screens, a number of putative MS1-interacting proteins. However, it is necessary to demonstrate their interaction with MS1 in planta. Such interactions can be visualised using fluorescently labelled fusion proteins by confocal microscopy and measuring FRET between interacting fluorescent proteins. However, such analyses are extremely difficulty given the low transient expression of these proteins, the autofluorescence of chlorophyll and the sensitivity of detection and limitations of the wavelengths that can be analysed. We propose to use a novel form of photon-counting detector based on STJs as a tool to detect and analyse the expression and complex formation of these MS1-interacting factors. STJs measure the energies of individual optical photons with high efficiency (up to 0.8 counts per incident photon) and extremely low internal background. The detector is cooled to cryogenic temperatures (~300 mK), but offers an advantage over competing photon counting systems that the entire fluorescent spectrum is detected on an event-by-event basis with temporal resolution of order microseconds. This means that the contributions of different fluorescent labels can be distinguished without the need for sequential filters and autofluorescence of microscope optics can be separately recorded and removed as a wavelength-dependent background source. The work will focus on 4 MS1-interacting proteins and will involve the preparation of fluorescent fusion proteins (eg CFP, YFP, GFP) regulated by the native, or CaMV35S, promoter. These will be integrated into plant material by bombardment or Agrobacterium transformation. Analysis of expression will be conducted by confocal microscopy and the STJ-based system. Co-bombardment and crossing between transgenic lines and STJ analysis, will be used to detect changes in the fluorescent emissions as a consequence of FRET.