Analysing GORK clustering for enhanced stomatal control

Stomata are pores that provide for gaseous exchange across the impermeable cuticle of plant leaves. They open and close to balance the requirement for CO2 entry for photosynthesis against the need to reduce the transpiration of water vapour and prevent leaf drying. Stomatal transpiration is at the centre of a crisis in water availability and crop production that is expected to unfold over the next 20-30 years: globally, agricultural water usage has increased 6-fold in the past 100 years, twice as fast as the human population, and is projected to double again before 2030. The bulk of water used in agriculture passes through the stomatal pore. Thus stomata represent an important target for manipulating crop performance. Significantly, stomatal responses are often delayed in the face of environmental fluctuations, especially of light. Improving water use efficiency (=amount of carbon fixed in photosynthesis/amount of water transpired) should be possible, without a cost to carbon assimilated in photosynthesis, if the speed of stomatal response can be enhanced.

Stomatal movements are driven by solute transport - and consequent uptake/loss of water - across the cell membrane of the guard cells which surround the stomatal pore. Guard cells harbour ion channel proteins to facilitate cation flux for stomatal movement. Uniquely, the opening (or gating) of one class of plant ion channels is also sensitive to external K+ concentration. These channels are found in guard cells of tobacco, Vicia and Arabidopsis, in the latter encoded solely by the GORK gene. Increasing K+ outside moderates channel opening in parallel with the equilibrium voltage for K+ and affects whole-cell conductance. These channels are the main pathway for K+ efflux during stomatal closure, but their K+-sensitivity constrains K+ flux capacity, notably at higher external K+. Estimates based on recent modelling suggests that stomatal closure could be accelerated 3-fold with only a moderate increase in the flux capacity of these channels.

The K+-sensitivity of the GORK channel is a property of the channel protein itself, which should facilitate manipulating K+ efflux capacity to accelerate stomatal closure. My laboratory has uncovered evidence that the K+-dependence of GORK gating is associated with its assembly in clusters. These assemblies require the the so-called 'voltage-sensor domains' (VSDs) of GORK to interact with one another. Movement of the VSDs is known to couple voltage to channel gating, so it is likely that interaction between VSDs provides a mechanism for cooperative self-regulation. I propose now to complete the analysis of GORK VSD interaction and the consequences for channel control and for stomatal movements. Regardless of the mechanism, it is clear that these discoveries offer the means to explore a unique and fundamental property of this class of K+ channels in plants and to manipulate channel activity, potentially enhancing the kinetics of stomatal closure and water use by the plant.

Gating of outward-rectifying K+ channels in plants, including the GORK channel of Arabidopsis guard cells, is uniquely sensitivity to extracellular K+. This sensitivity restricts substantially channel activity and K+ efflux during stomatal closure. Work from my laboratory has shown that the K+-dependence of gating is linked to GORK clustering. We have resolved one of the two, critical binding sites located on the voltage-sensor domain (VSD) of GORK and have confirmed that the second binding site at the N-terminus of the VSD. This project will resolve the second of the binding sites, and will utilize this knowledge to control clustering and modify gating. GORK is the primary K+ channel responsible for K+ efflux in the Arabidopsis guard cell, and makes this channel a important target for manipulations directed to accelerating stomatal closure and improving water use by the plant. Thus we will use knowledge of the binding sites to explore the potential for enhancing GORK activity.

Experiments will use in vivo and in vitro screens for binding and functional analysis. We will draw on the toolchest of vectors and expression methods developed in my laboratory for transient and stable transformations and mutant complementations. The work will be supported by structural modelling. Electrophysiological and related methods will be used quantify transport in vivo and after heterologous expression. We will assess stomatal responses to relate the effects of channel mutation on stomatal kinetics and their association with transpiration and carbon assimilation. We will use these data to parameterise physiological models and to test systems analysis in predicting enhanced stomatal behaviour and its consequences for water use efficiency. Thus, I fully expect new and exciting insights into guard cell transport and its coordination through targeted manipulations, much as our previous efforts provided hitherto unexpected insights through the mutational analysis.

This proposal is for research developing new concepts at the core of ideas emerging within the international transport, plant, and systems biology communities. The research will stimulate thinking around fundamental ion channel biophysics and membrane transport, plant growth and water relations, and it should strengthen biological and in silico methodologies for approaching crop engineering. Thus, the research is expected to benefit fundamental researchers and, in the longer-term agriculture and industry, through conceptual developments and targeted methods to improving plant water use efficiency and productivity. The research will feed into higher education training programmes through capacity building at the postgraduate and postdoctoral levels. Additional impact is proposed through public displays and the development of teaching resources building on the background work for this proposal. Finally the research will help guide future efforts in applications to agricultural/industrial systems. The applicant has established links with industrial/technology transfer partners and research institutes 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 Pathways.