Keeping in Touch: Visualising and Perturbing Photorespiratory Organelle Proximity in Plants

Abstract

Organelles within cells are typically depicted as isolated structures, but increasing evidence shows that they are interconnected via specific membrane contact sites (MCS). While MCS research in mammals and yeast is gaining importance, MCS are less explored in plants. Photorespiration is ametabolic process that occurs across three organelles in plants: chloroplasts, peroxisomes and mitochondria. Whereas this process is well studied on the molecular level, only little is known about the relevance of inter-organellar contacts. The aim of this study was to elucidate the significance of organellar interactions and to determine how MCS are linked to plant growth and performance with the photorespiratory organelles as a model. We addressed this question with three main experimental approaches: 1) quantifying the proximity between chloroplasts and peroxisomes under different photosynthetic conditions, 2) testing potential dynamic or irreversible reporter systems for organelle proximity, and 3) manipulating the spatial organisation of cells by introducing a synthetic tether construct. <br /> Previous reports evidenced physical associations and an increased interaction rate between the photorespiratory organelles in response to light in A. thaliana. We developed an automated high-throughput Python-based analysis pipeline for the quantification of organelle proximity. We used confocal z-stacks of cells with fluorescently labelled organelles and performed analyses in three model plant species. We were not able to replicate the findings of previous reports using manual image analysis or the Python-based analysis pipeline, potentially due to minor but critical changes in the experimental setup. <br /> Secondly, we tested potential fluorescence-based proximity reporters, based on Bimolecular Fluorescence Complementation (BiFC) or Forster Resonance Energy Transfer/Fluorescence Lifetime Imaging (FRET/FLIM).We successfully targeted the potential proximity reporters to the cytosolic face of the photorespiratory organelles. Using splitYFP-based proximity sensors, we found unspecific homogeneous organellar membrane labelling, whereas the investigation of organelle positioning revealed tethering between peroxisomes and chloroplasts. Moreover, we created an inducible 2in1 gateway vector system(pInd) to test the suitability of self-assembling GFP (saGFP) and to enable inducible expression in transgenic lines. Testing the expression of a saGFP-based proximity sensor between chloroplasts and peroxisomes transiently in N. tabacum, we found a peroxisomal membrane labelling with increased GFP signal at putative MCS. Using FRET/FLIM-based proximity sensors, controls mimicking 100% and no organellar interaction were established, while dynamic imaging did not reveal a decrease in fluorescence lifetime at putative MCS between chloroplasts and peroxisomes. <br /> The third approach involved introducing a synthetic tether to disturb the spatial organisation of the photorespiratory organelles. We were able to obtain transgenic A. thaliana lines showing curly leaves, impaired growth, an accelerated senescence, and a reduced high light tolerance including decreased anthocyanin accumulation. On cellular level, the overexpression of the synthetic tether resulted in the formation of spherical peroxisomal clusters, where mitochondrial structures also accumulated.