Using novel methods to investigate the phloem of higher plants
This thesis presents the results of an investigation into the phloem of higher plants. The functional unit of the phloem is the sieve element companion cell (SE:CC) complex. Following a period of surgical proteolysis the mature SE joins with others in a file to create a syncytial distribution network called the sieve tube (ST). This living conducting unit has a much-reduced protoplasm with the adjoining, ontogenically related, CC underwriting much of its basic functions. The SE is principally responsible for the long-distance transport of photosynthate from areas of production to areas of growth and storage. Additionally, this transport stream carries a myriad of dissolved growth factors, RNA and proteins that can act at discrete sites along the way. The mechanisms by which the phloem achieves this specificity amidst bulk flow remain to be resolved, but by accomplishing both the phloem coordinates concerted plant growth and development. Much of these details remain obscure as the phloem is recalcitrant to conventional study. The aim of this project was to tailor emerging technologies and develop new methodology specific to the study of the phloem. This thesis contains descriptions of the first ever super-resolution images taken of plants. This required a specific tissue processing schedule to be developed. The new insight afforded a greater understanding into the arrangement of viral movement proteins throughout the sieve plate and the parietal layer of the SE. Next a new technique for preserving fluorescence, antigenicity and fine structures is presented. This correlated imaging method allows vascular tissues to be examined by conventional light microscopy, super-resolution microscopy and the electron microscope. Through using this unifying method of sample preparation, it is possible to conduct an investigation from the tissue level to the nanometer scale. Finally, there is a survey of the SE parietal layer where a variety of the techniques discussed above are brought to bear upon the structures that survive the autolytic purge. It is hoped that these tools will find a wider use in the plant science community.