Synthesised in plants in small quantities, phytohormones are naturally occurring chemical messengers that play critical roles in regulating plant growth and development as well as triggering responses to external stimuli. The precise regulation of phytohormone biosynthesis, catabolism and transport is crucial to maintain these messengers’ concentration, allowing different parts of the plant to communicate and coordinate responses to changing environmental conditions. Understanding the biology of phytohormones has therefore developed to be an important field of study.
In this thesis, I focused on two specific hormones, Gibberellin (GA) and Salicylic acid (SA). I improved and characterised the next-generation Gibberellin Perception Sensors (GPS) based on the GPS1 in Chapter 3 and successfully designed and engineered a novel FRET-based biosensor for SA, Salicylic acid Sensor 1 (SalicS1) in Chapter 5. Using these biosensors, I examined the relationship between repatterning of the corresponding phytohormones and plant reprogramming under different stress conditions in Chapter 4 (GA) and Chapter 6 (SA). Aims of this thesis are summarised in the Figure below. These biosensors allowed the monitoring of changes in phytohormone levels with high spatial and temporal resolution and provided valuable insights into the complex interplay between phytohormones and environmental stimuli at the cellular level.
While nuclear-localised GPS1 (nlsGPS1) has been found to bind bioactive GA4 with high affinity and good signal-to-noise ratio, other GPS1 biosensor properties remained to be optimised and diversified. By modulating the interaction interface between the sensory domains AtGID1C and the truncated DELLA domain of AtGAI, we have successfully increased the *in vitro* reversibility of GPS1, and GA hypersensitivity phenotypes were reduced, resulting in GPS2. In my project, GPS2 was fully characterised. I further attempted to expand the range of GPS biosensor affinities through mutagenesis and by deploying higher affinity GID1 variants from other plant species. By altering the linkers between fluorescent proteins (FPs) and binding domain, I created GPS3 with a much-improved signal to noise ratio *in vitro* which allows accurate detection of smaller changes in GA levels particularly at low concentrations, although such properties were not observed *in planta*. I used nlsGPS1 to study the relationship between GA's redistribution and abiotic stresses, including nutrient deficiency, salinity stress, high sugar stress and osmotic stress.
I then focused on SA which is best known as a plant defence hormone and is also involved in increasing plant tolerance to several abiotic stresses. In this arm of my project, I developed a novel FRET based biosensor, SalicS1, to directly detect SA levels in live plants with unprecedented resolution. I screened SA receptors and their interaction partners from multiple species as ligand sensory domains. Combinations of various cyan-yellow FPs as FRET pairs and a set of linker variants connecting these four moieties generated single biosensor fusion proteins that were evaluated for the optimal SalicS1. SalicS1 response to SA was tested first *in vitro* after purification from yeast and then *in planta* in stable transgenic *Arabidopsis* lines, both in a dose dependent manner.
Using more low pH tolerant FPs will allow biosensors to be more widely subcellularly targeted, particularly in the acidic environments of the vacuole and apoplasm. My preliminary evidence indicate that some FRET pairs could lead to successful low pH-tolerant biosensors. Further engineering is needed to develop high signal-to-noise ratio low pH-tolerant biosensors (SalicSLowpH and GPSLowpH) to elucidate subcellular phytohormone distribution.
Nuclear localised SalicS1 (nlsSalicS1) were further used in studying the redistribution of SA levels under abiotic stresses. It revealed that SA were reduced in *Arabidopsis* seedlings roots. Other abiotic stresses, including low temperatures and salinity stress, were found to affect cellular SA levels, depending on the duration of exposure.
In conclusion, I fully characterised the GPS2 and created GPS3. I also engineered a novel FRET based SA sensor, SalicS1, to allow SA levels to be monitored in cellular scale *in vivo*. I attempted to design diversified sensor variants to be targeted to acidic subcellular environments. Taken together, my thesis has engineered and applied biosensors to advance our understanding of phytohormone redistribution at a high spatiotemporal resolution under stress conditions
