Stress in a crystal palace : using experimental micro-sensors to investigate light stress in Antarctic sea-ice diatoms

Antarctic sea-ice provides a highly structured yet dynamic environment for a diverse range of microbes. Ice-associated microalage, which survive in some of the most extreme environments on earth, contribute significantly to primary production in the Southern Ocean. Because the sea-ice matrix is highly spatially and temporally variable, microalgae must adapt to ranging physio chemical gradients and significant variation in solar irradiance. Light is the most influential driver of algal community dynamics, but due to variation in ice and snow thickness, light is both transient and inconstant. The ability of photoautotrophs to adapt to this ephemeral habitat requires highly efficient metabolic machinery to rapidly adapt to periods of high irradiance and extended periods of darkness. This thesis pairs microelectrodes, microfluidics and experimental sea-ice tanks with molecular mechanisms to investigate the physiological response of the model sea-ice diatom Fragilariopsis cylindrus to the complex variation in irradiance. Specifically, I examine how the three main seasonal changes in incident irradiance (darkness, sub-saturating and saturating) influences diatom physiology and how microelectrodes can be used to sense light-induced stress. In the first part of the thesis, I focus on proteomics, the molecular plasticity that enablesF. cylindrus to survive during extended darkness (Chapter 2). This is the first study to quantitatively analyse the whole cell proteome of a sea-ice diatom during prolonged darkness prior to re-illumination. I describe how this diatom utilises ancient alternative metabolic mechanisms to sustain essential metabolic processes in the dark to retain functionality of the photosynthetic apparatus. Chapter 3 details how F. cylindrus responds to modification in light intensity and spectral composition upon illumination after long-term darkness. I illustrate how extracellular production of hydrogen peroxide (H\\(_2\\)O\\(_2\\)), nitric oxide (NO) and glucose are produced in response to variation in spectral quality. I also examine the influence that exposure to different wavelength of light has on the physiology of F. cylindrus and demonstrate that the detection of extracellular products can be directly attributed to the photophysiological state of the cell. I suggest that the metabolic overflow of H|(_2\\)O\\(_2\\), NO and glucose is a useful proxy for the in situ detection of phototrophic stress of bottom-ice microbial communities. One of these metabolites, NO, is under-reported in microalgal research and has not been studied in ice-associated diatoms. In chapter 4, I investigate the mechanisms that enables NO production in F. cylindrus, and show that NO is nitrite-dependent; most likely via a nitrate reductase enzyme. Interestingly, NO production was abolished upon exposure to light, but could be induced in the light when normal photosynthetic electron flow was disrupted. Finally, the thesis applies the laboratory findings to an Antarctic field experiment, whereby in vivo extracellular metabolites produced by an under-ice microbial community were measured in response to rapid transitions in snow thickness. The results presented in chapter 5 illustrate that rapid changes in transmitted irradiance can impose a strong selective pressure on sea-ice taxa. The presence of a thin layer of snow (10 cm) positively influenced the under-ice algal biomass, whereas a rapid shift from thick snow (30 cm) to no snow caused significant oxidative stress in the under-ice community. I illustrate, for the first time, that extracellular 'stress' metabolites produced following environmental forcing can be measured in vivo and directly attributed to habitat modification. The results presented in this thesis provide a unique insight into plasticity of ice-associated diatoms with respect their capacity to survive long periods of darkness and to adapt to changes in irradiance. The production of extracellular metabolites was successfully measured using microelectrodes in both the laboratory and the field. The results indicate that under conditions of saturating irradiance that cause stress or disrupt normal photosynthetic electron flow, increased production of metabolites can be attributed to photophysiological state of the cell. The ability to measure in vivo overflow metabolism may provide an effective tool for real-time measurement of under-ice phototrophic health during periods of environmental change.