Engineering the GORK K+ channel to enhance stomatal kinetics

Lead Research Organisation: University of Glasgow
Department Name: College of Medical, Veterinary &Life Sci

Abstract

Stomata are pores that open and close to balance the requirement for CO2 entry to the leaf for photosynthesis against the need to reduce water loss via transpiration and prevent leaf drying. Stomata are 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 droughts of 2010-12 and 2018 cost UK farmers alone an estimated £1.2B and worldwide costs year-by-year are estimated in the hundreds of billions of pounds over the past five years. Thus stomata are an important target in efforts to improve crop performance, especially in the face of global climate change. Stomatal opening and closing are driven by solute and water transport of the guard cells which surround the stomatal pore. Our deep knowledge of these processes has made the guard cell one of the best-known plant cell models and gives real substance to prospects for engineering stomata to improve water use by crops.

In the natural environment light fluctuates, for example as clouds pass over. The stomata of most plants respond to light by opening the stomatal pore to increase CO2 access for photosynthesis, and they reduce the pore aperture when the light intensity drops and the demand for CO2 by photosynthesis declines. Photosynthesis generally tracks light fluctuations, but stomata are much slower to respond. The slower response of stomata can limit gas exchange and reduce carbon assimilation by photosynthesis when light intensity rises and lead to transpiration without corresponding assimilation when light intensity drops quickly. We and others have reasoned that assimilation, and consequently crop yields, could be enhanced concurrent with an decrease in water use by plants if the rates of stomatal movements could be better matched to variations in photosynthetic demand.

Recently, we found that accelerating ion flux in stomatal guard cells by introducing a synthetic, light-activated K+ channel, BLINK1, was sufficient to increase the biomass and reduce the associated water use by 2-fold in the model plant Arabidopsis. Furthermore, we have demonstrated that analogous gains are possible by altering the intrinsic controls on the activity of a K+ channel that occurs naturally in stomata and other plant cells. These findings demonstrate the potential of accelerating stomata as a strategy to enhance crop gains while conserving water and a second strategy based on the properties of a channel native to stomata.

We propose here an interlinked effort, combining our knowledge of native K+ channel regulation and of optogenetics in two distinct but related strategies. We will engineer native K+ channels for gains in water use efficiency and biomass yield and we will combine our knowledge of these channels with optogenetics to bring channel regulation under direct control by light. As a proof-of-principle, we will use Arabidopsis as a model that harbours K+ channels with orthologues in many crops. Additionally, we expect to develop and validate a new set of optogenetic tools and strategies based around modifications to the interactions of a known optogenetic photoswitch that will be widely applicable in plants. These aims dovetail with our longer-term interests in developing optogenetic approaches to bioengineering that integrate within processes native to the plant.

Technical Summary

We propose a concerted appraisal of two approaches to manipulating stomatal kinetics and its relevance to biomass gain and water use. Our aims are (1) to examine the potential for engineering the widely-distributed and naturally-occurring GORK K+ channel for these purposes through exploration of domains identified with inter-channel interactions in channel gating, and (2) to use knowledge of these domains in developing and applying new optogenetic strategies in vivo to accelerate stomatal kinetics for biomass and water use efficiency gains. A part of this work will develop and validate a new set of optogenetic tools based around separable components of the LOV2-J(alpha) photoswitch that are common to plants. We will build on our recent success with the light-activated K+ channel BLINK1 in Arabidopsis as a guide.

Both the practical and fundamental challenges will take advantage of targeted optogenetic expression within the leaf epidermis and, additionally, on the proven capacity for adjusting the gating controls of GORK to facilitate guard cell K+ flux and stomatal movements. Experiments will follow methodologies similar to those we have used successfully to date in molecular biology, protein-interaction and electrophysiological analysis, and in gas exchange and biomass studies. We will use extant knowledge of photoswitch variants and preliminary evidence for their deployment as separable components in developing and validating new optogenetic tools. In vivo, we will target expression to assess, in response to light, the impact and dynamic range of responses in stomatal guard cells that enhance their coupling to photosynthesis. We expect these studies to expand our fundamental understanding of stomatal mechanics, to establish a native K+ channel as a bona fide target for future efforts towards crop improvement, and to establish a new set of optogenetics tools with wide applicability in controlling protein-protein interactions.

Planned Impact

This proposal is for a synergy of fundamental research, albeit with practical implications in the longer term. The research builds on a core of ideas at the centre of the international plant photosynthesis and stomatal biology communities. It will stimulate thinking around strategies for enhancing crop yields and reducing agricultural water consumption, and it should inform methodologies for approaching crop engineering. In its essence, the research will yield basic insights into the physiology of stomatal guard cells as well as developing new methods in their engineering. Impacts will include the construction of a range of modified channel forms, both those without and with optogenetic control, and a new set of conceptual strategies for modifying membrane transporters native to the cell. In the long term, the research is expected to benefit practical research as well as informing agriculture and industry through the introduction of new technologies relevant to plant productivity and water use efficiency. The research will build capacity through higher education training programmes at the postgraduate and postdoctoral levels, yielding highly-trained researchers with broad expertise across molecular, cellular and whole-plant biology. Additional impact is proposed through public displays and the development of teaching resources building on the background work for this proposal. The research will help guide future efforts in applications to agricultural/industrial systems, and the applicants have established links with industrial/technology transfer partners and research institutes to take advantage of these developments. Details of these, and additional impacts will be found in the Case for Support and in the attached Impact Pathways.

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