Aquaporins
Aquaporins are well known as facilitators of membrane water transport in almost all organisms. Initially this was the central hypothesis for the study of aquaporin function in plants. Using different gene knock out techniques like antisense, RNAi, Virus Induced Gene Silencing or T-DNA insertions and comparing the resulting plants to controls, we could demonstrate that distinct aquaporins contribute to membrane water transport processes in plants. Among others, these concern root water uptake, cell elongation, plant growth and plant movement. In cooperation with two groups from Hebrew University of Jerusalem, we analyze water conducting aquaporins and their role in plant water transport in order to develop an optimized mathematical model for plant water uptake and optimize the water use efficiency of crops. By this, our basic knowledge about aquaporins will find an application in farming.
Other plant aquaporins do not facilitate water membrane transport and we have consequently searched for other substances than water that show a facilitated membrane transport in the presence of these aquaporins. Results from physiological studies lead us to the conclusion that the above mentioned aquaporin species facilitate CO2 membrane transport and it appears that for many gaseous substances the molecular membrane transport mechanism is not completely understood. In particular, the transport of gases like CO2, O2 or NH3 and the regulation of membrane water transport in correlation to other substances are under investigation. Current data indicate for the significance of aquaporins for gas transport and regard their function to many physiological processes like photosynthesis, respiration or growth. It is interesting to note that similar proteins were found also in humans and bacteria, which give us the chance to cooperate with non-botany scientists. Consequently besides plant physiologists, some of our main collaborators work in human science, microbiology or biophysics.
As main goals, we try to characterize the gas transport function of specific aquaporins, their regulation, their structure, the molecular mechanism and their implication in physiology not only in plants.
Aquaporins in synthetic model systems
The Kaldenhoff group has an expertise in functional characterization of membrane pore proteins named Aquaporins. Besides their activity as membrane water transport facilitators, we have evidence for a physiological significant increase in gas transport rates by some of these proteins. In collaboration with Frank Bernhard (Goethe University Frankfurt, Germany) and coworkers we have used an in vitro transcription/translation system that was established by the Frankfurt group to express plant aquaporins to milligram amounts. Reconstitution of the plant aquaporins as well as that of human aquaporins in liposomes and functional analysis showed that the extracellular synthesized proteins were active in the synthetic system. The group of Wolfgang Meier (Basel University, Swiss) has established a procedure to incorporate membrane proteins like aquaporins in synthetic material. These block-copolymers could form membrane-like structures and vesicles. If plant aquaporins produced and purified in the Kaldenhoff lab were integrated in block-copolymers and form vesicles their function resembles that in cellular systems (collaboration with J. Zilles and M. Kumar, ; University of Illinois, USA). Our current approaches are aiming to analyze aquaporin facilitated CO2 transport by a moving micro pH electrode in a setup, where two chambers are separated through the synthetic material (block co-polymer). We envisage an application where CO2 transport facilitating aquaporins in synthetic material provide a gas selective filter.
Plant Parasites
Parasitic plants could lead to complete crop failure. The analysis of molecular reactions during the parasitic infection process and throughout the plant – plant interaction provided the basis for strategies to reduce events of a successful parasite infestation. We have identified host proteins (e.g. attAGP) that are induced by attachment of the parasitic plant Cuscuta. By introduction of a corresponding RNAi construct that was transferred to the host tomato using virus induced gene silencing, we could demonstrate a linear correlation between attAGP expression and successful parasite attachment. On the other hand, we have identified several proteins that are expressed upon infection by the parasitic plant. One of these represents a proteinase. Identification of a specific inhibitor of the proteinase function and expression of the encoding gene in E. coli provided us with a substance that could be used as a Cuscuta repellent. Application and further use of this inhibitor is subjected to a European Patent claimed by TU. Transgenic plants expressing the inhibitor together with an extracellular targeting sequence were found to be resistant. Field trials of products developed on the basis of the inhibitor to date are conducted in collaboration with local institutes in Cuscuta-affected regions in Africa, China, Israel and Palestine.
Similarity in the attachment mechanisms of the parasitic plant species Cuscuta, Striga and Orobanchae lead to a joint project with a Palestine and an Israeli group, which aims to develop an applicable strategy of plant defense towards these plant parasites. Besides the above mentioned proteins these benefit from a protein exchange between host and parasite. This mechanism will be used to target host derived proteins that inhibit parasite specific metabolism to the parasite and thus inhibit parasite growth.
