“Turning A New Leaf” Development of Spectroscopic Protocols to Study Plant Health

Improved methods to monitor plant health may be pivotal for future protection of crops from disease and environmental stressors. Epicuticular waxes, and macronutrients (K+ and Ca2+) hold important physiological functions within plants, and are therefore, excellent markers of plant health. This thesis has developed new spectroscopic analytical methods to directly image changes in epicuticular waxes and Ca2+ and K+ on plant leaves, for future application to monitor plant health

Synchrotron Macro ATR-FTIR Microspectroscopy for High Resolution Chemical Mapping of Single Cells

Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy has been used widely for probing the molecular properties of materials. Coupling a synchrotron infrared (IR) beam to an ATR element using a high numerical aperture (NA) microscope objective enhances the spatial resolution, relative to transmission or transflectance microspectroscopy, by a factor proportional to the refractive index (n) of the ATR element. This work presents the development of the synchrotron macro ATR-FTIR microspectroscopy at Australian Synchrotron Infrared Microspectroscopy (IRM) Beamline, and demonstrates that high quality FTIR chemical maps of single cells and tissues can be achieved at an enhanced spatial resolution. The so-called “hybrid” macro ATR-FTIR device was developed by modifying the cantilever arm of a standard Bruker macro ATR-FTIR unit to accept germanium (Ge) ATR elements with different facet sizes (i.e. 1 mm, 250 μm and 100 μm in diameter) suitable for different types of sample surfaces. We demonstrated the capability of the technique for high-resolution single cell analysis of malaria-infected red blood cells, individual neurons in a brain tissue and cellular structures of a Eucalyptus leaf. The ability to measure a range of samples from soft membranes to hard cell wall structures demonstrates the potential of the technique for high-resolution chemical mapping across a broad range of applications in biology, medicine and environmental science

“Wax On, Wax Off”: In Vivo Imaging of Plant Physiology and Disease with Fourier Transform Infrared Reflectance Microspectroscopy

Analysis of the epicuticular wax layer on the surface of plant leaves can provide a unique window into plant physiology and responses to environmental stimuli. Well-established analytical methodologies can quantify epicuticular wax composition, yet few methods are capable of imaging wax distribution in situ or in vivo. Here, the first report of Fourier transform infrared (FTIR) reflectance spectroscopic imaging as a non-destructive, in situ, method to investigate variation in epicuticular wax distribution at 25 µm spatial resolution is presented. The authors demonstrate in vivo imaging of alterations in epicuticular waxes during leaf development and in situ imaging during plant disease or exposure to environmental stressors. It is envisaged that this new analytical capability will enable in vivo studies of plants to provide insights into how the physiology of plants and crops respond to environmental stresses such as disease, soil contamination, drought, soil acidity, and climate change

Synchrotron X-ray fluorescence microscopy-enabled elemental mapping illuminates the “battle for nutrients” between plant and pathogen

Metal homeostasis is integral to normal plant growth and development. During plant-pathogen interactions, the host and pathogen compete for the same nutrients, potentially impacting on nutritional homeostasis. Our knowledge of outcome of the interaction in terms of metal homeostasis is still limited. Here, we employed X-ray fluorescence microscopy (XFM) beamline at the Australian Synchrotron to visualise and analyse the fate of nutrients in wheat leaves infected with Pyrenophora tritici-repentis, a necrotrophic fungal pathogen. We sought to (i) evaluate the utility of XFM for sub-micron mapping of essential mineral nutrients and ii) examine the spatiotemporal impact of a pathogen on nutrient distribution in leaves. XFM maps of K, Ca, Fe, Cu, Mn, and Zn revealed substantial hyperaccumulation within, and depletion around, the infected region relative to uninfected control samples. Fungal mycelia were visualised as threadlike structures in the Cu and Zn maps. The hyperaccumulation of Mn in the lesion and localised depletion in asymptomatic tissue surrounding the lesion was unexpected. Similarly, Ca accumulated at the periphery of symptomatic region and as micro-accumulations aligning with fungal mycelia. Collectively, our results highlight that XFM imaging provides capability for high resolution mapping of elements to probe nutrient distribution in hydrated diseased leaves in situ