Harnessing Synthetic Biology for Retrograde Signalling in Plants.

Abstract

Plants rely on precise communication between organelles and the nucleus to regulate gene expression-based stress responses, enabling acclimation to changing environments that impact photosynthesis and respiration. A major pathway in this communication is retrograde signaling, where signals from organelles such as chloroplasts inform the nucleus of cellular status. Among these signals, reactive oxygen species (ROS) play a central role. However, their reactive and overlapping nature complicates the study of individual ROS effects. Hydrogen peroxide (H2O2) is a key retrograde signal, functioning both as a damaging molecule under oxidative stress and as a trigger for protective gene expression. Yet, studying H2O2 directly remains difficult due to its overlap with other signals. This thesis aims to develop a synthetic toolkit for investigating the direct role of H2O2 and eventually enable construction of customizable signaling circuits to enhance plant stress response specificity and sensitivity. By engineering modular components that mimic or respond to H2O2 signaling, this work lays the foundation for precise probing of redox biology and future improvements in crop resilience. The first stage focuses on establishing chloroplast outer envelope targeting for synthetic sensing. Given the chloroplast's role in environmental sensing, I hypothesized that positioning synthetic modules at its surface would enable fast, spatially relevant responses. I screened ten targeting domains fused to mCherry in Arabidopsis protoplasts, identifying domains capable of specific outer envelope localization for use in later toolkit development. Building on this, I investigated aquaporins as H2O2 transporters in modulating chloroplast-derived H2O2 flux. I used AtTIP1;1, previously shown to transport H2O2, to explore how altering aquaporin-mediated flux affects retrograde signaling. Fusing AtTIP1;1 to the selected targeting domains, I confirmed that these modifications did not impair H2O2 transport. AlphaFold2 modeling showed intact pore structures, and yeast assays confirmed comparable transport efficiency between native and engineered AtTIP1;1. GFP-tagged constructs confirmed membrane localization in yeast. Additionally, I generated AtTIP1;1 overexpression lines in both wild-type and knockout Arabidopsis backgrounds to provide resources for studying increased H2O2 sensitivity in planta. The final part of this work developed a synthetic H2O2-responsive cleavage system based on the transcription factor ANAC017. Under oxidative stress, ANAC017 is cleaved from the endoplasmic reticulum membrane, releasing it to activate gene expression. I adapted this natural cleavage event into a synthetic tool by fusing ANAC017's cleavage site to mCherry, tethered to the chloroplast outer envelope. Protoplast expression and exogenous H2O2 treatment revealed possible cleavage and reporter release, indicating preserved functionality in this synthetic context. In parallel, I evaluated two orthogonal proteases (TVMVP and TEVP) for conditional cleavage of synthetic constructs. Co-expression in protoplasts showed efficient cleavage with minimal cross-reactivity, supporting their use in logic-based synthetic circuits. Together, this work establishes a modular toolkit combining chloroplast targeting, engineered H2O2 transporters, and cleavage-based response modules. These tools enable new strategies for dissecting and rewiring retrograde signaling, advancing both the understanding of H2O2's role in plant stress signaling and the development of synthetic biology applications in crops. Show more