ABA transport at the nexus of nutrient deficiency and water stress in plants

Agricultural food production heavily relies on mineral fertilization. To date, the global annual use of phosphorous (P) fertilizer alone stands at 43 Mio tons. The European Commission has classified P as a 'critical raw material' and the UK is 100% reliant on import. Adding the negative impacts on environment and human health, the current rate of P use is clearly not sustainable. Developing ways to grow crops with less P input is therefore paramount for national and global food security.

Due to their sedentary lifestyle, plants are risk-adverse and prepare for the worst-case scenario when they perceive environmental challenges. They have mechanisms to detect a decrease in P supply and immediately react. Up-regulation of high-affinity transport and root branching enhances soil 'mining' while down-regulation of growth and energy consumption safeguards internal resources.. If we can delay the latter and optimize the former, we have an opportunity to close the gap between apparent and potential yield. However, to put these ideas into practice through crop breeding or genome editing, we need a precise understanding of molecular signalling pathways that underpin early responses of plants to P deficiency. Considering water shortage and climate change, we also need to know whether these pathways interact with those mediating responses to osmotic stress imposed by drought or salt intrusion.

We recently discovered that knockout of a gene called NPF4.2 in Arabidopsis thaliana completely abolishes early main root inhibition in low P. NPF4.2 encodes a transporter for abscisic acid (ABA) and is located on the vacuolar membrane of cells located in the central vasculature of the root. While the roots of npf4.2 mutants continue to grow in low P they can still be inhibited by other nutrient deficiencies or by salt. These findings not only highlight an entirely new role of the 'stress hormone' ABA for P-deficiency responses but also point to new role of ABA transporters for endowing the pathway with specificity.
The aim of this project is to precisely map differences and convergence of the signalling pathways that inhibit root growth in response to low-P and osmotic stress and to position NPF4.2 in this network. To this end we will take advantage of the advanced tools available for A. thaliana. We will employ recently developed technology for in-vivo ABA-imaging and single-cell transcriptomics alongside reverse genetics and protein biochemistry.

The proposed work programme has three parts. Work package 1 will deliver spatial maps of stress-evoked ABA signatures in roots, which will be overlaid with response patterns of related signals such as Ca2+, ROS and pH, and with spatial root transcriptomes. Work package 2 will tell us how NP4.2 shapes the signal signatures, in collaboration with other ABA-transporters and with enzymes that mobilize ABA-storage forms. This work package will also identify the relationship between NPF4.2 and previously identified components of the low-P signalling pathway such as ferroxidases and CLE peptides. The last work package will produce information on how the NPF4.2 protein is regulated. Candidate targets will be selected from the transcriptomics studies with particular emphasis on interactions with low-P induced members CIPK and CBL gene families. CBL/CIPK regulons are already known for activating membrane transporters in a Ca2+-dependent manner thereby effectuating responses nutrient and salt stress. However, a role in regulating ABA transport would be entirely novel.

The expected outcomes will provide a fundamental science base for the development of 'smart' crops combining improved resource use with robustness against abiotic stress. We will identify key points in the signalling pathways that will allow us to de-couple or connect different signal inputs and response outputs. This research will therefore open offers new opportunities for precision agriculture in different environment scenarios.

Technical summery
Plant hormones guide the growth and development of plants. Changes in their spatial-dynamic signatures provide means for rapid adaptation to fluctuating conditions in the environment. Precise information on the location and regulation of auxin transporters has been instrumental for the generation of quantitative models that explain how plants re-direct auxin fluxes to trigger developmental adaptations. Such precise information is still missing for ABA. Recent research has implied a plasma-membrane located protein in ABA transport from vascular storage sites into guard cells.

We have now discovered that a tonoplast-localised ABA transporter in the root stele (NPF4.2) of Arabidopsis thaliana is required for main root inhibition by low P, but not by salt or external ABA. This highlights not only a new function of ABA in low-P responses but also suggests that ABA transporters endow ABA-signalling pathways with specificity for different stress factors.

The proposed work programme has three parts. We will apply genetically encoded FRET-based ABA-sensors to monitor ABA signatures under low P and salt stress in roots. In combination with sensors for ROS and Ca, and with single-cell transcriptomics, this work will pinpoint similarities and differences in origins and targets of the signalling pathways. We will then test whether and how the signatures are modulated in npf4.2 mutants and use a genetics approach to position NPF4.2 within current models of P- and ABA-signalling. Finally, we will identify regulators of NPF4.2 in the yeast split-ubiquitin system alongside BiFC, pull-downs and phosphorylation assays.

The work will generate fundamentally new insights into where and how ABA-signalling pathways differentiate and converge. This knowledge will provide new molecular targets for controlling stress responses of crops either individually or in combination for improved performance under reduced P and/or water availability.