Final Report Summary - CELLSEC (Studying plant cell secretion and membrane trafficking) The work developed during this project consisted mainly of two independent but inter-related research topics: (A) The establishment of the role of SNARE proteins during polarized growth of pollen tubes. (B) The establishment of the mechanisms regulation ion influx and the pollen tube plasma membrane and their role upon membrane secretion. The main goals of both topics were achieved with original data submitted in three publications (one printed, one in press, one under final preparation). Part A Part A of the project was devoted to the study of pollen specific SNAREs, proteins which are involved in the docking and fusion of secretory vesicles with the plasma membrane thus modulating membrane secretion. Little data is available on the action mode of these proteins in plant cells thus their relevance for our research. The pollen specific syntaxis SYP 124 and SYP 125 (Qa SNARES) were used and the interaction studies were carried out with GTPase Rab11 and phosphatidylinositol-4-monophosphate 5-kinase 4(PIP4K5). We showed that Arabidopsis thaliana syntaxins are involved in the process of polarized vesicle secretion which is essential for tip growth. The results presented suggest that in normal growing pollen tubes, a net exocytic flow occurs in the flanks of the tube apex mediated by SYP124 and SYP125. The specific distribution of SYPs at the plasma membrane is affected by changes in Ca2+ levels in agreement with the importance of this ion for exocytosis. Apical growth and the specific localization of SYPs were affected by regulators of membrane secretion (Ca2+, PIP5K4 and Rab11) but competition with a dominant negative mutants affected only SYP distribution. These data thus suggest that syntaxins alone do not provide the level of specificity required for apical growth but additional signalling and functional mechanisms are required. Two manuscripts resulted from this part of the work. Part B: Along with docking of vesicles, another important element dictating fusion of vesicles and thus secretion of growth components, is the level of calcium ions in this region. It is known that extracellular Ca2+ influx is crucial for this process but little is known about the protein channels that regulate this influx. Previous research from the group has suggested the involvement of cyclic nucleotides during this process so here we investigated the role of CNGC channels in pollen tube growth. The Cyclic Nucleotide Gated Channels - CNGCs - chosen for this study were nr 7 and 8. CNGC7 and CNGC8 are pollen specific and were identified in Arabidopsis thaliana; the sites of expression of these genes were confirmed by GUS reporter. T-DNA insertion lines for these genes were used and analysed regarding vegetative growth and pollen assays. Pollen tube morphology and elongation was evaluated in vitro and in vivo and fertility was investigated using classical genetic approaches. Functional redundancy was suspected due to pollen lethality of double homozygous plants. To confirm this, the double homozygous mutant lines were complemented with either CNGC7 or CNGC8 GFP reporter constructs. The complemented lines were then analyzed regarding vegetative morphology and reproductive ability (pollen tube growth and guidance, fertilization, number of seeds, transmission to progeny). The expression of GFP in these lines was also used to study CNGC7 and CNGC8 subcellular localization. One manuscript (under final steps of preparation) resulted from this part of the project. Potential impact and socioeconomic impact Considering the research area - sexual plant reproduction - the social and ethical impact will be considerably reduced. The data obtained will be of interest particularly to the scientific community and for agro-industrial companies since this project was designed to shed light on the fertilisation mechanisms which ultimately dictate seed production.
