MicroX-ray absorption near edge structure tomography reveals cell-specific changes of Zn ligands in leaves of turnip yellow mosaic virus infected plants

As many metals are essential for plants, excess or deficiency of them or alteration of their uptake, translocation, sequestration or physiological use can have severe consequences for plant growth and fitness. Therefore, investigating the distribution and speciation of metals in tissues is essential to understand plant physiology. We present a method based on non-destructive Synchrotron X-ray microtomography combined with microspectroscopy for studying metal distribution and speciation in plant tissues. By using the Maia detector system and the high flux of the undulator beam at the P06 beamline of the PETRA III synchrotron (at DESY), it was possible to record micro X-ray Absorption Near Edge Structure (mu XANES) for every voxel of a tomogram. The metal co-ordination in regions of interest within the tissue samples could be determined by comparing the XANES with spectra of relevant reference compounds. Metal distribution and coordination were measured in shock frozen hydrated plant leaves in a cryostream, avoiding sample preparation artefacts like liquid cell content redistribution that occurs with other preparation methods, unequal distribution of stains in staining assays, sample degradation by beam damage and thawing, etc. A spatial resolution of 5 mu m was selected, which is sufficient to resolve all leaf tissues (epidermis, palisade mesophyll, spongy mesophyll, veins), larger cells and biomineralization hotspots. As an application example, we studied the effect of infection with Turnip Yellow Mosaic Virus (TYMV) on the Zn distribution and the Zn speciation in duplicates of Noccaea ochroleucum. This non-accumulator plant grown with 100 mu M Zn had enough metal to allow collecting significant spectroscopic data. We found that the TYMV infected samples formed biomineralization crystallites, showing strong spectroscopic similarity to Zn silicate.