Sounding the all clear: investigating how and why plant cells deplete the stress hormone ABA

Drought stress is a major environmental stress that impairs crop production worldwide, but tolerance to drought and other stresses is a plant trait that varies greatly among cultivars and crop species. Tolerance of a plant to a stress condition is the result of coordinated action of many processes and thus rational improvement of crop stress tolerance will require a detailed and sophisticated understanding of plant stress biology. The plant hormone abscisic acid (ABA) plays a key role in controlling responses to environmental stress conditions like drought stress. ABA is also an important regulator in plant growth and development regulating processes such as seed dormancy and root growth. Plants have to adjust ABA levels constantly in order to match physiology and development to ever changing environmental conditions. It is known that ABA accumulated in cells during a stress condition is a temporary event that is followed by ABA depletion, maintaining a tight temporal regulation of ABA responses. Although three biochemical activities - breakdown, conversion to a storage form, and export from the cell - are known to lower cellular ABA levels, a quantitative understanding of how these combine to determine ABA depletion rates in plant cells remains elusive. A deeper understanding of these mechanisms and how they are controlled is important in improving the ability of crop plants to tune their development to suit their environment.

The proposed research project aims to uncover dynamic ABA patterns in root cells and to understand how these dynamic patterns relate to ABA-dependent root development. The knowledge will be expanded to identify the mechanisms determining how root development responds to environmental nitrate availability. Biosensors that report concentrations of ABA by directly binding to ABA in cells have been used to measure dynamic ABA patterns in Arabidopsis thaliana, a reference plant for molecular biology research. Using time-course, microscopic imaging of growing Arabidopsis roots expressing ABA concentration and uptake sensors (ABACUS), we have already observed that ABA depletion rates vary in space and time. We now aim to understand how several biochemical activities combine to articulate ABA levels into dynamic patterns appropriate for a given environmental condition. Spatial and temporal depletion of ABA will be studied in detail using Arabidopsis mutants that are affected in ABA depletion activities, thus revealing the impact of each activity on ABA depletion rates. Root growth phenotypes of these mutants will be examined concurrently with ABA levels. Linking the maps of ABA depletion rates to corresponding root growth phenotypes will provide detailed hypotheses regarding how ABA impacts plant development. For example, ABA is thought to play a role in attuning root architecture to the levels of nitrate in the environment. Imaging ABACUS in root cells responding to nitrate availability will be carried out to pinpoint the specific cell-type and timing of ABA accumulations that control root nitrate responses. This type of detailed knowledge can then guide targeted interventions into crop plants to improve agricultural resilience to environmental stress.

Another main objective of the project is to engineer next generation ABACUS sensors for improved high sensitivity visualisation of ABA dynamic patterning in roots and other plant tissues. In addition, a new biosensor - Sensor of Abscisic Acid-Glucose Ester (SAGE) - will be developed as a powerful tool to address the question of where and when ABA-glucose ester pools, inactive 'storage' forms of ABA, are important in development and environmental responses.

Broadening the knowledge of spatio-temporal patterning of ABA will be important in understanding the mechanisms underlying physiological and developmental adjustments during environmental stresses that can prevent significant crop losses.

Abscisic acid (ABA) is a key plant hormone that directs drought stress tolerance and developmental processes such as germination. Recently developed ABA concentration and uptake sensors (ABACUS) have been successfully used to begin quantifying the timing and locale of ABA accumulation and depletion in growing Arabidopsis roots with confocal time-course imaging.

Objective A. Visualising ABA during genetic and environmental perturbations using ABACUS
ABA depletion is a spatially and temporally regulated process that has a functional relevance to root development. The impact of several biochemical mechanisms on cell-specific ABA depletion rates will be investigated by mapping ABA depletion in mutants affected in ABA catabolism, conjugation, and export. These spatiotemporal maps of ABA depletion will be linked with root growth phenotypes of the ABA depletion mutants to understand the functional role of specific ABA depletion mechanisms. One of the functions of ABA is to attune root architecture to nitrate availability in the environment. Nitrate-mediated repatterning of ABA in roots will be studied using mutants of ABA deconjugation enzymes. These studies promise to reveal not only the rules of how ABA is depleted in a cell-specific manner in roots, but also the functional consequences of ABA patterning for root development.

Objective B. Biosensor engineering
Next generation ABACUS will be engineered to have high-affinity for ABA, high signal-to-noise ratio, high stability of expression and low impact on endogenous signalling pathways to overcome the limitations associated with the current ABACUS. A new sensor, Sensor of Abscisic Acid-Glucose Ester (SAGE) will also be developed as a powerful tool to address the question of where and when ABA-GE pools are important in development and environmental responses. These biosensors can be used to bring ABA spatiotemporal maps, as well as their functional relevance, into sharper focus.

This project's fundamental aim is to understand how cellular concentrations of the key stress hormone abscisic acid (ABA) are dynamically patterned in plant roots and how these dynamic patterns relate to root developmental decisions, particularly in response to environmental perturbations such as changes in the supply of nutrients. One of the challenges in plant developmental biology is to understand how plant cells and tissues integrate signals from the environment with endogenous signals and then respond to these signals with growth decisions that attune development to the environment. Plant hormones serve as signal integrators and master regulators of developmental decisions. Thus, deeper understanding of plant hormones can help predict and adapt the responses of plant development to a changing climate.
In summary, we foresee the following impacts from this project and our research:
1. UK growers: understanding spatiotemporal patterning of ABA could lead to economic and competitive benefits for UK farmers facing challenges in crop development or physiology related to ABA.

The PI will continue existing collaborations with UK growers such as G's and participate in CambPlants Hub, which acts as a forum for breeding companies and local farmers groups (http://www.cambplants.group.cam.ac.uk/).

2. The Public: this type of basic biological question is excellent for engaging public interest in science - many people are surprised to learn that plants 'listen' to their environments and sometimes respond by changing their body plans. The team will participate in public engagement.
SLCU has a very active public engagement program (http://www.slcu.cam.ac.uk/outreach/outreach.html). For example, the PI participated in the Cambridge Science Festival by presenting a station during the SLCU scientific discovery trail. We will participate in at least two such outreach events each year.
3. Agricultural Breeders: The fundamental research findings of this project could generate clear leads for plant breeders working towards developing more stress tolerant crops. Such crops will both enhance quality of life for farmers and increased global food security and public health. Applied research topics relating to this project include plant breeding for drought tolerance, optimising seed dormancy, and tailoring root architecture in challenging environments.

The PI is already in communication with collaborators at the National Institute for Agricultural Botany (http://www.niab.com/) that could potentially translate the research findings into crop plants. The PI also attended a convening on 'Plant Communication' in April 2016 at the Gates Foundation in Seattle and made contacts with people making positive impacts on agriculture in developing nations. Furthermore, the PI will participate in a new initiative entitled Cambridge Global Food Security to discover ways of bringing the project's scientific findings and technological tools to bear in tackling the great challenge of ensuring global food security in the face of diminished resources and a changing climate.

4. Scientific community: the impacts of this project will be passed on to the scientific community through publication, presentations at meetings and conferences, continued open dialogs with world-wide colleagues, and deposition of research tools at public repositories.

The PI and postdoc will attend international and domestic meetings to present the findings and technological innovations of this project. Due to the emerging nature of the field of high-resolution hormone quantification in vivo, we will write review articles and methods/protocols to increase the exposure of the field within traditional plant biology (multiple invited reviews and protocols are already in preparation). Mutant and biosensor lines will be deposited at public repositories such as Addgene and ABRC as was done for previous biosensors.