ABACUS1s have a positive ratio change with high signal-to-noise ratio but poor sensitivity for endogenous ABA 14. Ligand-induced conformational changes in sensory domains alter the relative positions of the FPs, which can be detected by exciting the donor and measuring a change in relative acceptor and donor emissions, hereafter referred to as emission ratio change.ĪBAleons are negative ratio change biosensors that are sensitive to endogenous ABA concentrations, but have poor signal-to-noise ratios (small emission ratio change) 13, 20. The orientation and distance between these FPs determine the transfer of excitation energy via FRET from a donor FP to an acceptor FP 21. In ABAleons and ABACUS1 biosensors, ABA sensory domains are connected by linkers to a pair of fluorescent proteins 13, 14 (FP) (Supplementary Fig. Therefore, we engineered next-generation ABA biosensors and deployed them to dissect cellular ABA dynamics and mobilization in response to foliar humidity stress, and to establish a systemic role for ABA in maintaining local root growth in response to a distant shoot stress. However, existing ABA FRET biosensors, ABAleons and Abscisic Acid Concentration and Uptake Sensors 1 (ABACUS1s) 13, 14, 20 lack the full complement of strengths in terms of the signal-to-noise ratio or affinity required to easily quantify ABA. Direct ABA FRET biosensors 13, 14 that do not require additional signalling components have broad application potential beyond ABA quantification in plant cells and subcellular compartments for example, in ABA synthesizing pathogenic fungi 17, in human granulocytes where ABA is a cytokine 18, or in extracts from organisms where genetic modification is difficult using purified protein in vitro 19. Such biosensors are powerful tools to quantify metabolites in vivo at high spatiotemporal resolution 11, including phytohormones under changing environmental conditions 12, 13, 14, 15, 16. The availability of sensitive reporters, particularly Förster resonance energy transfer (FRET) biosensors, for hormones, second messengers and metabolism is revolutionizing plant development, signalling and photosynthesis research 11. The sites of ABA biosynthesis, metabolism and translocation are the subject of intensive research, but progress has been hampered by limitations in tools to quantify accumulation and depletion of ABA on a tissue/cellular scale where regulatory decisions controlling ABA dynamics are made 1, 10. Although a molecular mechanism remains elusive, it has been proposed that ABA, acting as a dehydration signal, could be coordinating these root growth responses 5, 9. Interestingly, leaf water loss can cause changes in root growth responses and architecture: increasing transpiration genetically or through increased airflow produces larger root systems in Arabidopsis 5 and low relative humidity (RH) can promote root growth in many species 6, 7, 8. When roots experience low-water stress, for example, ABA closes the microscopic pores on leaves (stomata) to limit systemic water loss 2, 3, 4. Abscisic acid (ABA) is a phytohormone that accumulates systemically under various local water stresses to coordinate responses over a complex and often-large morphology 1. Plant decision-making is distributed rather than centrally coordinated, but to survive and overcome stresses such as lack of water, responses must also be systemically coordinated.
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