88 Phase Separation Of Arabidopsis NCP Proteins in Regulating Plant Thermotolerance

Faculty Mentor: Chan Yul Yoo (Biological Sciences, University of Utah)

Abstract

Global climate change has led to a steady rise in air temperatures since pre-industrial times, making plant thermotolerance a necessity for survival. Heat stress negatively impacts plant growth and resilience by interfering with physiological processes. To mitigate these effects, plants have evolved protective mechanisms that preserve cellular function and important structures, including organelles like chloroplasts. As the site of photosynthesis and a key driver of plant growth and development, the chloroplast plays a central role in sensing environmental stress and initiating adaptive responses to protect the plant as a whole. NUCLEAR CONTROL OF PEP ACTIVITY (NCP) is a chloroplast- and nucleus-localized protein that plays roles in two major photomorphogenic developmental processes: hypocotyl growth inhibition and chloroplast biogenesis in Arabidopsis thaliana (Arabidopsis).

Sequence analysis revealed that NCP contains two predicted intrinsically disordered regions (IDRs), but the functions of the domains remain unknown. Given that IDRs often drive liquid-liquid phase separation (LLPS), I hypothesized that the IDRs of NCP enable LLPS and contribute to heat stress adaptation. NCP localized in the chloroplast may undergo LLPS in response to heat, forming condensates that act as signals to the chloroplast to initiate stress response pathways for plant thermotolerance. To test this, I performed molecular cloning to develop green fluorescent protein (GFP) tagged constructs, including GFP alone as a negative control, GFP-FUS as a positive control, and [latex]\text{GFP-NCP}_{48}[/latex]. GFP alone is not known to show LLPS capabilities and GFP-FUS is known to form liquid droplets, making for ideal controls. After performing protein purification to express and isolate recombinant GFP-tagged NCP protein, I performed phase separation assays in vitro and used microscopy to capture the results. This study investigates whether NCP can undergo LLPS in vitro under conditions that mimic heat stress and explores the potential relevance of this behavior in the broader context of plant thermotolerance. In vitro assays revealed that GFP-NCP undergoes LLPS and forms liquid-like droplets in the presence of PEG as a molecular crowding agent. Notably, heat stress significantly enhanced droplet formation. I propose that heat-induced condensation of NCP promotes chloroplast-mediated protective responses and enhances plant survival under heat stress conditions. Together, this study demonstrates that NCP undergoes LLPS in vitro and suggests a potential role for NCP condensates in protecting chloroplast structure and function under heat stress. The chloroplast will then signal to the nucleus to upregulate the translation of heat shock proteins (HSPs) to protect important and heat-sensitive proteins. Understanding the phase separation properties of NCP lays the groundwork for future studies aimed at elucidating its physiological function in plants. Including ongoing studies of the IDRs of NCP for determining the domain(s) driving LLPS capabilities. The insights gained from this study contribute to a broader understanding of biomolecular condensation in plant stress responses and inform ongoing efforts to engineer crops with enhanced thermotolerance.

Please read the full paper, under the same title, in the Marriott Library digital archives.

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