Fe determination and distribution in plant samples by XRF methods

Iron (Fe) deficiency is one of the major agricultural problems which highly affects the production of cultivated plants, especially in calcareous soils. Therefore, studying Fe uptake and homeostasis in plants is extremely important to develop new agricultural practices aiming at increasing Fe availability in soil for plant nutrition. However, determining Fe concentration and distribution in plants by XRF methods is not always simple, especially in Fe-deficient plants, where Fe concentration can be even below 1 ppm. In this study we investigated the potentiality of different XRF techniques to detect, quantify and map Fe distribution in different plant tissues. In particular, we used synchrotron micro-XRF to map quantitatively Fe in leaf and root (with a confocal geometry) tissues. For comparison, Fe maps were also acquired by using laboratory micro-XRF instruments, both commercial and in-house built. Advantages and limitations, in comparison to synchrotron-based methods will be discussed. In particular, Fe distribution was investigated in leaves of cucumber plants (Cucumis sativus L.) grown in different conditions (-Fe, +Fe, +Fe-chelates) whit three laboratory micro-XRF spectrometers, two polychromatic polycapillary focused micro-XRF and a mono-chromatic micro-XRF developed by the XMI Group at Ghent University. The data showed that, as expected, synchrotron XRF measurements gave better results in a shorter time. However, XRF laboratory instruments gave good results, which can be still useful for agronomic studies. While the mono-chromatic source gave a better signal to noise ratio, thus providing better spectra for quantitative evaluation even at low ppm levels, the two commercial instruments provided better maps (also thanks to the smaller beam size), which means more accurate information about Fe distribution throughout leaves tissues. TXRF is another XRF method very useful to quantify Fe in plant samples, even below 1 ppm level. This technique is particularly suitable to determine Fe concentration in xylem sap and plant tissues with a very limited sample preparation, thus reducing the costs and the artefacts caused by an extensive sample manipulation. Examples from plants grown in symbiosis with plant-growth-promoting-bacteria (PGPB) or cultivated with innovative agronomic practices will be presented. In particular, roots and leaves of cucumber were analysed as powder suspensions while samples of xylem sap, extracted from olive trees, were analysed directly. An increase of Fe concentration was observed in both root and leaves of cucumber treated with PGPB. On the contrary, no variation of Fe concentration was found in the xylem sap of olive trees. In conclusion, all these examples are intended to show the major advantages and limitations of using XRF techniques to study Fe homeostasis in plants and suggest the best XRF analytical approach to be adopted according to the specific case study.