In a plant cell, the apoplast is the extracellular space filled with fluid that contains necessary materials and enzymes for root growth and development. I hypothesize that the apoplastic space is a reservoir of calcium ions, which plays a crucial role in helping to regulate cell wall dynamics. Apoplastic Ca2+ crosslinks with pectin to form gel-like structures that influence cell wall rigidity. There are also a variety of channels that permit the movement of apoplastic calcium into the cytoplasm, such as through cytoplasmic Ca2+ bursts, leading to cell expansion. Understanding cell wall dynamics involving calcium movement can provide insights about how calcium can act as a messenger between the soil microbiome and root cells. Currently, there are no tools to track Ca2+ dynamics in the apoplast, since existing indicators are optimized for pH 6.0-7.0 at low concentrations (20-100 nM in cytoplasm), while the apoplast is acidic (pH ~5.2) and contains high Ca2+ levels (1-10 mM or higher). This project aims to engineer a new Ca2+ biosensor that is able to function within the acidic environment of the apoplast and explore its relationship with the structure of the cell wall. Using Förster Resonance Energy Transfer (FRET) as the mechanism to engineer the indicator, this sensor incorporates acidic-tolerant fluorescent proteins and a low-affinity Ca2+ binding domain to enhance its performance. To optimize the binding domain and linker for FRET, we will create a library of candidates to be expressed in E. coli. The performance of the sensor will then have to be confirmed and characterized in order to ensure its stability in the apoplast. This indicator can elucidate the role of Ca2+ in plant root and microbes interaction and how they distinguish different types of symbiotic relationships. Furthermore, this new indicator has potential applications in other acidic regions within the cell to track calcium dynamics, such as in the lysosome or vacuole, and it can also be tested across different plant species to explore variations in calcium requirements and distribution.
