Characterising diel-specific nuclear microenvironments using spectral phasor analysis in Arabidopsis thaliana

Abstract

The circadian clock is an internal timekeeper that anticipates and responds to environmental diel cycles. At the cellular level, the clock involves gene activation and repression in concert with environmental cues such as light, temperature and potentially humidity1 . This impacts plant growth, including apical root growth, and overall plant health. Circadian gene expression is often studied through in vitro and in planta mutant gene expression, however, the clock may be studied indirectly by characterising spectral profiles over time. These spectral profiles, or spectral microenvironments, can be investigated using hyperspectral microscopy and spectral phasor analysis. Hyperspectral analysis combines microscopy cell imaging with spectroscopy spectrum detection, allowing for niche profiling of nuclear behaviour and the natural fluctuations across diel phases in planta. Here, the Arabidopsis thaliana root model was used to showcase diel shifts of nuclei spectra across two diel phases, pre-dusk (end of day, ZT-12) and pre-dawn (end of night, ZT-0). First, a plant preparation protocol was created so seedlings could remain in their growing medium during microscopy, termed the “GPI plating technique” for seedlings Germinated at the agar Plate Interface. These seedlings grew comparably to their “traditionally grown” counterparts and had an approximate 115% decrease in damage signatures alongside morphological assessment, suggesting this technique is a less destructive and minimally invasive plating protocol. Next, the fluorescent stain outlining the nucleus was assessed, with SybrGreen (I) demonstrably less invasive to the cell with higher fluorescent signal than the initially chosen stain Acridine Orange. SYBR Green (I) characterisation also presented consistent, diel-phase specific shifts in spectral widths. Across three A. thaliana root cell types, nuclei of seedlings entrained to pre-dawn phase (ZT-0) showed significantly wider widths within cell category, with collated data showing an 8.1nm width increase. Wavelength characterisation was not found to be significant. These findings could later be complimented with circadian gene expression experiments, to validate both diel and circadian spectral profiles. Overall, this thesis demonstrated that A. thaliana root nuclei have a consistent spectral width profile that is diel-, or time-, specific. These findings were possible using the minimally disruptive yet highly sensitive analysis techniques of Hyperspectral analysis imaged through Spectral Phasor Analysis.

Date of Award	2023
Original language	English
Awarding Institution	Western Sydney University
Supervisor	Mark Jones (Supervisor)