Physiology and localisation of pollen ion transporter

After landing on a receptive stigma, pollen grains are activating all cellular processes necessary for growing a pollen tube that penetrates the stigma tissue and transports the gametes to the ovary where finally fertilisation occurs. Due to their simple organisation, pollen tubes serve as a model organism for investigating tip growth processes. Germinating and growing pollen tubes generate an electrical field around themselves with a typical pattern of in- and effluxes of K+ , H+ , Cl - , and Ca2+, which have been measured by ion-sensitive vibrating probes. It is expected that these ion currents are generated by specific ion transporters in the plasma membrane that show a local distribution and therefore cause locally restricted ion currents. This detailed physiological data were mainly obtained from lily (Lilium longiflorum) pollen grains and tubes which did not seem suitable for molecular biology studies compared to Arabidopsis thaliana pollen. Recently, the applicant`s group measured and identified K+ channels and H+ ATPases from lily pollen grains and tubes. The electrophysiological properties of these ion transporters will be characterised by patch-clamp techniques in a heterologous expression system, namely ion transporter-deficient mutants of yeast (Saccharomyces cerevisiea) and in Xenopus oocytes. Fluorescence-tagged (GFP, FlAsH) ion transporter allow their localisation and observation of their turn-over in the plasma membrane. In addition, the constructs will be re-cloned with pollen-specific promoters for transient as well as stable transformation of lily pollen. Particle bombardment of lily pollen grains allows a transient expression of the desired, fluorescence-tagged ion transporter to monitor its localisation during pollen tube growth. The distribution of the respective ion transporter (K+ channel or H+ ATPase) in the plasma membrane can then be compared with the well-characterised electrical current pattern and the effects of genetically engineered/modified ion transporters on pollen tube growth can be investigated. The planned stable expression of the ion transporter in the pollen grain and tube needs the development of reliable protocols for transformation and regeneration of lily plants from calli, but then allows simultaneous measurements of the ion current pattern and the localisation of the responsible transporter. In this project already identified ion transporters will be characterised in yeast mutants and in Xenopus oocytes, localised in transgenic cells and most important, the localisation in the growing pollen tube will allow a correlation with the current pattern monitored in growing pollen tubes and genetically modified ion transporters may allow to investigate their specific role in pollen physiology. In future, every ion transporter identified from pollen can be measured with the same set of techniques and vector/plasmid material needing only re-cloning of the transporter- of-interest. This project closes a gap between the physiologically well-characterised model pollen of lily (monocots) and the genetically-characterised pollen of Arabidopsis and Nicotiana (dicots). Additionally, successful transformation of lily plants will also be important for ornamental horticulture.

In this project basic principles for the generation of an electrical field around growing pollen tubes were investigated: the function and localization of two main components, the plasma membrane H+ ATPase and the K+ channel. Both ion transporters were identified and sequenced in lily pollen, a physiological model system for pollen tube growth, and their physiological role during polar pollen tube growth was studied.A successful fertilization is an essential prerequisite for the production of seeds and fruits for the growing human population. During this process, the male pollen grain transports the sperm cells to the female ovaries. After falling onto a stigma, the pollen grain generates a pollen tube that grows through the pistil tissue towards the egg cell and there, releases the sperm cells for fertilization. Pollen tubes contain a kind of navigation system which helps to find the egg cell. This navigation system consists of a Ca2+ gradient in the tube tip and a special pattern of plasma membrane ion transporters which generate an electrical current pattern or an electrical field around the tube determining the tubes growth direction. If the navigation system fails, pollen tubes miss the egg cells which directly results in low crop yields. An important role during osmoregulation and regulation of the turgor pressure was shown for the PM H+ ATPase. This enables an optimal adaption of the pollen tube to varying osmotic conditions and allows continuous growth towards the egg cell without bursting. The 14-3-3 proteins involved in the regulation of the PM H+ ATPase were also identified in lily pollen. Currently, the molecular details of this interaction are studied using recombinant-produced lily pollen 14-3-3 proteins and a regulatory part of the PM H+ ATPase. Surprisingly, the K+ channel (LilKT1) was not detectable in the lily pollen tube plasma membrane. This phenomenon was studied in more detail in a yeast and a plant expression system. It could be shown that only in plant expression systems the LilKT1 is partly localized at the plasma membrane. Therefore, the physiological role of this K+ channel is still not solved.