A protein scaffold essential for K+ transport and stomatal control

This proposal builds on the discovery in this laboratory of an unusual SNARE protein complex that is essential for K+ channel regulation in Arabidopsis, and of our recent finding of its impact on K+ uptake by the roots and plant growth. Because the SNARE, SYP121, is already known to have roles in pathogen defence and cell volume responses meidated by the plant hormone abscisic acid (ABA), these findings offer the first substantive evidence that physically links the protein complex to actions in abiotic stress, mineral nutrition, water use efficiency (defined as the amount of dry matter produced per unit of water transpired through stomata) and cell expansion in the plant. 'Closing the circle' of K+ channel control, its contributions to K+ assimilation, water flux and cell volume control in the growing plant is expected to yield substantive insights in future efforts towards 'hardening' and development of water-effcient crops. In general, eukaryotic cells engage SNARE proteins as part of a well-defined mechanism for traffic of membrane vesicles, proteins and soluble cargo between compartments within the cell. Work from this laboratory first identified genes encoding two plasma membrane SNAREs from tobacco and Arabidopsis, and demonstrated their association with the ABA signalling cascade leading stomata to close and reduce water loss from the plant. Much is known about these cellular signals as well as the mechanisms they engage in regulating transmembrane ion flux. By contrast, we have little knowledge of the mechanisms coordinating ion transport with cellular volume and membrane surface in guard cells. Indeed, how plant cells regulate transport of osmotically-active solutes (especially of K+) in parallel with cell volume, whether reversible as in guard cells or during irreversible expansive growth, remains a matter of considerable debate. Knowing how transport is coordinated with changes in cell surface area will be of great fundament importance to understanding cell volume control in plants generally, and is likely to have practical relevance in our ability to manipulate the growth and homeostasis of plants, for example in altering stomatal controls to improve water use efficiency of crops. Our working hypothesis is that SNARE-K+ channel interaction serves as a molecular governor, analogous to the mechanical invention of James Watt, to coordinate channel-mediated uptake of the osmotically-active K+ ion with cell expansion We want to explore the molecular mechanics of the interaction, its implications for such a governor model in cell volume control and, equally, we want to examine its potential for applications in agriculture. Our immediate goals are (1) to explore the dynamics of the protein partners and their interactions, (2) to analyse their impact on ion transport and its coordination with membrane traffic, and (3) to assess their importance for stomatal function and water use by plants under stress.

This proposal builds on our discovery of an unusual SNARE protein complex that is essential for K+ channel regulation in Arabidopsis, and of our recent finding of its impact on K+ uptake and plant growth. In general, eukaryotic cells engage SNARE proteins as part of a well-defined mechanism for traffic of membrane vesicles, proteins and soluble cargo between compartments within the cell. Our discovery of a SNARE-K+ channel complex in plants, and evidence of its unusual role in controlling K+ uptake as well as facilitating cell expansion, provides the very first opportunity to explore how such interactions regulate transport of osmotically-active solutes in parallel with cell expansion in plants. Knowing how transport is coordinated with changes in cell surface area will be of great importance to understanding cell volume control in plants generally, and is likely to have practical relevance in our ability to manipulate the growth and homeostasis, for example in altering stomatal controls to improve water use efficiency of crops. Our working hypothesis is that SNARE-K+ channel interaction serves as a molecular 'governor' to coordinate channel-mediated uptake of the osmotically-active K+ ion with cell expansion. The immediate goals are to analyse the molecular basis for the interaction, its implications for cell volume control and, equally, to examine its potential for applications in agriculture. These studies will make use of site-directed mutagenesis, multi-component protein interaction analysis, electrophysiology and single-cell imaging technologies to identify the protein motifs essential for SNARE-K+ channel interaction and to characterise their role(s) in K+ transport control in parallel with changes in cell volume.

This proposal is for fundamental research developing new concepts at the core of ideas emerging within the international plant cell biology community. The research should stimulate thinking about these topics and help facilitate a paradigm shift in approach. These studies will also extend our recent development of a novel 'bridge' assay for high-throughput molecular interaction analysis. Thus, the research is expected to benefit fundamental researchers as well as industry through conceptual developments as well as the introduction of new technologies for the analysis of multicomponent systems. The research will feed into higher education training programmes through research training at the postgraduate and postdoctoral levels. Finally it will help guide future efforts in applications to agricultural/industrial systems. MRB has established links with industrial/technology transfer partners (Agrisera, Dualsystems, Plant Bioscience) and research institutes (SCRI and JIC) to take advantage of these developments. Further details of these, and additional impacts will be found in Part 1 of the Case for Support and in the attached Impact Plan.