Improved sample preparation for fruits allowed histochemistry and X-ray microscopy to reveal conserved iron hotspots

Mature fleshy fruits are the most challenging plant organs to study due to their high water and flavonoid content. We aim to develop a sample preparation protocol to analyze fleshy fruits with histochemical and X-ray based methods. By vacuum freeze drying and decolorization with fixatives, we were able to apply histochemical Perls staining to seamlessly localize iron in fruits. We screened the fruits to reveal possible conserved iron accumulation sites. This approach showed iron preferentially accumulated in fruit vascular tissues; at subcellular level, in the cell walls of vasculature and other tissues, and inside the epidermal cells of polyphenol-rich fruits. Furthermore, iron accummulated in the endosperm of tomato seeds, indicating a role in germination. X-ray microscopy of chemically untreated fruit slices showed similar iron distributions, indicating chemicals used in the protocol did not significantly mobilized the metals. This has been the first systematic study to extend histochemistry and X-ray fluorescence to obtain spatial information from mature fleshy fruits. This advancement in methodology may facilitate fruit research, potentially contributing to food security in the long run. ### Competing Interest Statement The authors have declared no competing interest.