Pollen-pistil communication in flowering plants: a role for endocytosis?

Reproduction success in flowering plants is unequivocally determined by the species-specific communication events between pollen and pistil resulting in fertilisation, and the development of viable seeds ultimately. Upon pollination, only own pollen will be accepted and continue their journey through the pistil whereas all other 'foreign' pollen will be rejected. After pollination, pollen grains produce a tube needed for the accurate delivery of sperm cells to the female embryo sacs deep down in the pistil ovary, harbouring the egg cells that are waiting to be fertilised. These so-called pollen tubes show polarized growth, a type of cell growth which is characterised by extension at the growing apical domain only. Signals provided by the different female pistil tissues guide these pollen tubes in the right direction over long distances in a relatively short time. How growing pollen tubes perceive these signals & translate them into a (species-specific) growth response is currently not known. Two processes of membrane trafficking at the apex characterize this polarized growth in pollen tubes: exocytosis and endocytosis. In other eukaryote cell types, including in plants, the process of endocytosis has been demonstrated to play an important role in cell and tissue signalling. However, a role for endocytosis in intra and inter-specific pollen tube communication with the surrounding pistil tissues has not been established yet.

This PhD-project includes a wide range of state-of-the-art research techniques: from protein biochemistry, molecular biology, genetics, microscopy and imaging analysis, to mathematical modelling. In collaboration with Dr Emyr Lloyd-Evans, the successful candidate will identify and functionally characterise pollen and pistil-proteins that are taken up by pollen tube endocytosis, and establish a link between the endocytic activity found during the polarised tip growth with pollen tube growth efficiency in vivo.

During Rotation project 1, the student will work in my lab and s/he will obtain intensive training in the more general & basic tools used in molecular biology, protein analysis (isolation, recombinant expression, PAGE and Western Blotting), working with plants and confocal microscopy (in collaboration with Dr Walter Dewitte). We will generate T-DNA constructs used for the development of transgenic plant lines, including Arabidopsis thaliana, containing novel pollen-specific organelle GFP marker proteins.

For Rotation Project 2, the student will work with the Dr E Loyd-Evans at Cardiff University to adapt their mammalian endo-lysosomal purification methodologies for use in plant cells, i.e., pollen tubes. For the first part of this rotation the student will determine and then refine the best separation strategy of protein fractions of interest, e.g. ultracentrifugation of (sucrose) gradients, analysis will include fluorescence microscopy, electron microscopy and western blotting. Secondly, the student will determine what influence endosomal Ca2+ homeostasis has on pollen tube growth. The EL-E lab has developed a number of techniques to determine the impact of localised changes in Ca2+ on a multitude of mammalian lysosome proteins and ultimately their cellular function. These techniques will be adapted for use in plant cells where Ca2+ tip gradients are known to be essential for pollen tube growth.