Biochemie pflanzlicher Membranproteine
Proteinanalytik, Pflanzenphysiologie, Pflanzenhormone
Auxin is a major plant hormone that has to be properly transported and redistributed throughout the plant at all stages of a plant’s life cycle. The proteins involved in controlling and directing auxin distribution are thus key determinants of many aspects of plant growth, development and physiological responses. The PINs are a family of integral membrane proteins that play a crucial role in the export of auxin from cells, but the actual mode of action by which they mediate or control auxin transport is unknown. As PINs show no significant homology to other proteins, the lack of experimental data on their molecular properties hampers progress in this field.
The research activities of my “plant membrane protein biochemistry” group aims to address this gap in knowledge by using biochemical approaches to elucidate some of the basic structural features of PIN proteins, such as membrane topology, N-glycosylation, inter/intra-molecular disulfide bonds and oligomerisation, the last of which is a feature that is integral to the mechanism of action of many membrane transporters and receptors. Such data should reveal some of the molecular features of PIN proteins, which will aid in the interpretation of past and future studies on the involvement of PINs in plant auxin distribution.
Establishing methods for investigating Arabidopsis PIN proteins
The PIN family consists of eight members, PIN1 to PIN8. Each PIN has a specific expression zone in the plant and specific roles in a variety of processes. We have decided to cover at least 5 to 6 members in our analysis (PIN1, PIN2, PIN3, PIN4 and PIN7, possibly PIN8) to obtain a comprehensive understanding of these proteins, as it may be the differences between these closely related members that will provide the most insights into the connection between structure and function.
In contrast to most other studies which rely heavily or almost exclusively on the use of recombinant fusion or tagged versions of the PINs (e.g. the very widely used fluorescent PIN-GFP constructs), we prefer to detect the proteins using antibodies raised against the endogenous proteins. This allows the investigation of naturally occurring endogenous proteins in wild-type plants, and avoids the problems associated with unusual or atypical behavior of many fusion proteins and genetically modified lines. In addition, we have recently also managed to detect endogenous PID protein, a kinase that is known to control PIN proteins.
Fig.1 GUS-stained Arabidopsis seedling showing the expression zone of a protein
Examining possible post-translational modifications of PIN proteins
From the pattern of bands seen in western blot analysis, it is apparent that PIN proteins can be modified post-translationally and that such modifications differ between the various PIN proteins. These modifications can be altered under certain conditions, e.g. following treatment with inhibitors of various signalling pathways. We are currently investigating the nature of such modifications.
Fig.2 Proteins separated by SDS-PAGE and stained with Coomassie Blue (left) or Ponceau S (right)
Investigating the gravitropic response in Arabidopsis CK2 mutants
We have recently discovered that Arabidopsis CK2 mutants have a faster and more accurate root gravitropic response than wild-type plants. In this project, we are presently investigating the reason for this, including analysis of changes in PIN and PID protein expression and the involvement of the ethylene signalling pathway.
Fig.3 Arabidopsis root tip
Fig.4 Interactions between transmembrane helices of membrane proteins in the plasma membrane
Fig.5 Roots of the CK2 mutant responding to a gravity stimulus
The significance of light/dark transitions on plant growth and behaviour
As plants grow in cycles of light and darkness, almost every aspect of their growth and behaviour is affected to some extent by the presence or absence of light and/or by circadian rhythms. We are interested in how plants react to light as they develop, i.e. from the first few days following germination compared to older stages.
Abas, L., Luschnig, C. (2010) Maximum yields of microsomal-type membranes from small amounts of plant material without requiring ultracentrifugation. Anal. Biochem. 401, 217-227.
Huang, F., Zago, M.K., Abas, L., van Marion, A., Galvan-Ampudia, C.S., Offringa, R. (2010) Phosphorylation of conserved PIN motifs directs Arabidopsis PIN1 polarity and auxin transport. Plant Cell 22, 1129-1142.
Marhavý, P., Bielach, A., Abas, L., Abuzeineh, A., Tanaka, H., Pařezová, M., Petrášek, J., Kleine-Vehn, J. & Benkova, E. (2011) Cytokinin modulates endocytic trafficking of PIN1 auxin efflux carrier to control plant organogenesis. Development 21, 796-804.
Marquès-Bueno, M., Moreno-Romero, J., Abas, L., De Michele, R, and Martínez, M.C. (2011) A dominant negative mutant of protein kinase CK2 exhibits altered auxin responses in Arabidopsis. Plant Journal 67, 169-180.
Malenica, N., Abas, L., Benjamins, R., Kitakura, S., Sigmund, H.F., Jun, K.S., Hauser, M.-T., Friml, J., and Luschnig, C.,MODULATOR OF PIN genes control steady-state levels of Arabidopsis PIN proteins. The Plant Journal, 2007, 51, 537-550.
Abas, L., Benjamins, R., Malenica, N., Paciorek, T., Wisniewska, J., Moulinier-Anzola, J., Sieberer, T., Friml, J. and Luschnig, C. Intracellular trafficking and proteolysis of the Arabidopsis auxin efflux facilitator PIN2 are involved in root gravitropism. Nat. Cell Biol., 2006, 8: 249-256.
Petrásek, J., Mravec, J., Bouchard, R., Blakeslee, J.J., Abas, L., Seifertová, D., Wisniewska, J., Tadele, Z., Kubes, M., Covanová, M., Dhonukshe, P., Skupa, P., Benková, E., Perry, L., Krecek, P., Lee, O.R., Fink, G.R., Geisler, M., Murphy, A.S., Luschnig, C., Zazímalová, E., and Friml, J. PIN proteins perform a rate-limiting function in cellular auxin efflux, Science, 2006, 312: 914-918.
In recent years our group published together with the following groups:
Christian Luschnig, DAGZ/BOKU; Vienna, Austria
Eva Benkova, VIB; Gent, Belgium
Pierre Hilson, VIB; Gent, Belgium
Jiri Friml, VIB; Gent, Belgium
Remko Offringa, University of Leiden; Leiden, The Netherlands
Carmen Martinez, Universitat Autonoma de Barcelona; Barcelona, Spain